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PGK1 Monoclonal Antibody (22C5D8)

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PGK1 Antibody — Figure 2. Rio1 localizes to kinetochores and promotes replication fork velocity/stability at the rDNA. ( A ) To probe the localization of Rio1 to kinetochores, we submitted a strain endogenously expressing 6Myc-RIO1 and its untagged parent to anti-Myc based chromatin immunoprecipitation, followed by microarray hybridization (ChIP-chip). The hybridization profiles (orange) at CEN5 and CEN10 are shown. ( B ) To corroborate the localization of Rio1 to kinetochores by microscopy, we isolated the nuclei from G1 cells (all 16 kinetochores are concentrated near the spindle pole) and then spread, washed and submitted them to indirect IF analysis. We identified Rio1 at both the rDNA (Nop1 as the rDNA/nucleolar marker) and at kinetochores (Ndc80 as the marker). DAPI strongly dyes the chromosomes but not the nucleolus as the latter is highly enriched with proteins, preventing DAPI from staining the rDNA. Scale bar = 5 mum. ( C ) To evaluate the contribution of Rio1 activity to chromosome replication we analyzed replication intermediates at the rDNA array and at negative control ARS305 in HU-treated RIO1-AID, P ADH1 -OsTIR1-9Myc cells (next named RIO1-AID ) that contained or were depleted of Rio1 activity. The RIO1-AID strain was grown exponentially (expon), then synchronized in G1 with alpha-factor (alphaF) and finally released into the cell cycle using 200 mM HU-containing YPD medium supplemented with 500 muM of auxin or mock solution. Samples were collected at the indicated

PGK1 Antibody — Figure 1. Uptake of beta-alanine via the Gap1 permease causes Rag/Gtr-dependent TORC1 activation without increasing the internal pools of amino acids. ( A ) Top. Wild-type (w-t) and gap1Delta cells were grown on Gluc Pro medium and [ 14 C]-beta-ala (0.5 mM) was added to the medium before measurement of the incorporated radioactivity at various times. Bottom. w-t cells were grown for 4 days on solid minimal medium without any N source or with GABA (0.5 mM) or beta-ala (0.5 mM) as sole N source. ( B ) gap1Delta cells expressing, from plasmids, a gene encoding GFP-fused Gap1, Gap1(K9R-K16R), or Gap1-126 were grown on Gal Pro medium. Glucose was added for 30 min to stop Gap1 neosynthesis prior to addition of beta-ala (0.5 mM) for 4 min. Crude cell extracts were prepared and immunoblotted with anti-GFP and anti-Pma1 antibodies. ( C ) Fluorescence microscopy analysis of the cells in 1B. Cells were grown on Gal Pro medium. Glucose was added for 1.5 hr to stop Gap1 neosynthesis, and beta-ala (0.5 mM) was added for 30 min or 1 hr. CMAC staining (blue) was used to highlight the vacuole. ( D ) Top. w-t and gap1Delta cells expressing HA-Npr1 from a plasmid were grown on Gluc Pro medium. Cells were collected before and at various times after addition of beta-ala (0.5 mM). Crude extracts were prepared and immunoblotted with anti-HA and anti-Pgk antibodies. Bottom. Same as in the top panel, except that w-t cells were collected before and 4 min after addition of beta-ala (0.5 mM). Rap was ad

PGK1 Antibody — Published figure using PGK1 monoclonal antibody (Product # 459250)

PGK1 Antibody — Published figure using PGK1 monoclonal antibody (Product # 459250)

PGK1 Antibody — Figure 4 Deletion of RSC1 in an Rco1-degron background results in a partial suppression of the small STE11 cryptic transcript. (A) Northern blot of RNA extracted from indicated degron strains at 0, 40, 80, and 160 minutes post-degron induction. Blots were probed with amplicons from the 3 and apos; region of STE11 or from SCR1 (loading control). Cryptic transcripts are indicated with dashed arrow and full-length transcripts by solid arrow. Blots are representative of 3 biological repeats. (B) Densitometry analysis of bands from northern blots in (A). Data is shown as percent change in band volume at the 160 minute time point compared to time zero (Y-axis) for each transcript in each strain (X-axis). Image Quant (GE Biosciences) was used to quantitate band density on the northern blot. All STE11 transcripts were normalized to the loading control, SCR1 . Error bars represent the average deviation of three biological repeats. (C) Western blot of protein extracted from degron strains at indicated time post degron induction. Tagged Rco1 protein was detected with anti-FLAG antibody. The same blots were also probed with anti-Pgk1 as a loading control. Blots are representative of 3 biological repeats. (D-F) ChIP followed by qPCR using primers corresponding to the STE11 ORF was performed with antibodies against acetylated H4. The X-axis indicates the mid-position of each probe set used in the PCR (base pairs). The Y-axis represents percent maximum change of the ChIP product at 40 minutes (

PGK1 Antibody — Figure 5 Deletion of BDF1 in an Rco1-degron strain suppresses the small STE11 cryptic transcript. (A) Northern blot of RNA extracted from indicated degron strains at 0, 40, 80, and 160 minutes post-degron induction. Blots were probed with amplicons from the 3 and apos; region of STE11 , FLO8 or SCR1 (loading control). Cryptic transcripts are indicated with dashed arrow and full-length transcripts by solid arrow. (B) Western blot of protein extracted from degron strains at indicated time post degron induction. Rco1 protein was detected with anti-FLAG antibody. The same blots were also probed with anti-Pgk1 as a loading control. (C) ChIP followed by qPCR using primers corresponding to the STE11 ORF was performed with antibody against acetylated H4. The X-axis indicates the mid-position of each probe used in the PCR (base pairs). The Y-axis represents percent maximum change of the ChIP product at 160 minutes versus 0 minutes post degron induction for each strain. Error bars indicate standard deviation (n = 3). (D) Northern blot of RNA extracted from swr1 Delta, Rco1 degron strain at 0, 40, 80, and 160 minutes post degron induction. Blots were probed with amplicons from the 3 and apos; region of STE11 or SCR1 (loading control). Cryptic transcripts are indicated with dashed arrow and full-length transcripts by solid arrow. (E) ChIP followed by qPCR using primers corresponding to the STE11 ORF was performed with antibody against the SAGA subunit, Ada2. The X-axis indicates the mid-position

PGK1 Antibody — Published figure using PGK1 monoclonal antibody (Product # 459250)

PGK1 Antibody — Figure 7. TORC1 activation in response to increased cytosolic H + involves the Pma1 H + -ATPase. ( A ) GAL1p-PMA1 pma2Delta cells transformed with the YCp(Sc)PMA1, YEp(Np)PMA4 882Ochre , or pFL36 (empty) plasmid (-) were grown for 3 days on solid medium with Pro as sole N source and Gal or Gluc as carbon source. ( B ) GAL1p-PMA1 pma2Delta cells expressing (Sc)Pma1 or (Np)Pma4 882Ochre from a plasmid (as in A) were grown on Gluc Pro medium in a microplate reader for 40 hr. Data points represent averages of the OD at 660 nm of two biological replicates; error bars represent SD. ( C ) Strains and growth conditions as in B, except that the strains were also transformed with a plasmid (pHl-U) expressing pHluorin . The cytosolic pH was monitored during growth with or without addition, starting at 1 min, of FCCP (20 uM) for various times. ( D ) Left. Cells as in B but also expressing HA-Npr1 from a plasmid were grown on Gluc NH 4 + medium. After a shift to Gluc Pro medium for 3 hr, [ 14 C]-beta-ala (0.1 or 1 mM) was added to the medium before measuring the incorporated radioactivity at various times. Right. Strains and growth conditions as in left. Uptake via Gap1 of [ 14 C]-beta-ala (provided at 0.1 mM to ( Sc ) PMA1 -expressing cells and at 1 mM to (Np)PMA4 882Ochre -expressing cells) was measured before and after glucose starvation for 5 min or after addition of FCCP (20 uM) or its solvent alone (0.2% EtOH) for 5 min. Plotted values represent percentages of initial Gap1 activity,

PGK1 Antibody — FIGURE 6: Atg1-DD shows severe autophagy defect. (A) Pho8Delta60 assay monitoring bulk autophagy was carried out in rich media (YPD) or nitrogen-starvation (SD-N) conditions. Samples were normalized to the activity of cells rescued with wild type under autophagy-inducing conditions. Results are average of three independent experiments. Error bars: SD. (B) mCherry-Atg8 processing assay showing autophagy defect in Atg1 DD mutant. Atg1 DD cells endogenously expressing mCherry-Atg8 were grown to log phase in rich media (YPD) and autophagy was induced by nitrogen starvation (SD-N). WT, wild type. mCherry (conjugated: 43 kDa; free: 29 kDa) was detected by Western blot. (C) Western blot showing expression of wild-type and double-aspartate mutant Atg1. Loading control: PGK1. (D) SIM of endogenous Atg1-DD mutant and mutant rescued with wild-type Atg1. Quantitation of distribution of structures found in mutant and rescue cell lines. Quantitation of wild-type and rescue strains was done via SIM; quantitation of the DD mutant was done in wide-field microscopy after confirming the existence of only punctate structures via structured illumination. Wild type n = 98; rescue n = 52; DD mutant n = 168. Scale bar: 1 mum.

PGK1 Antibody — Published figure using PGK1 monoclonal antibody (Product # 459250)

PGK1 Antibody — Published figure using PGK1 monoclonal antibody (Product # 459250)

PGK1 Antibody — Published figure using PGK1 monoclonal antibody (Product # 459250)

PGK1 Antibody — Published figure using PGK1 monoclonal antibody (Product # 459250)

PGK1 Antibody — Published figure using PGK1 monoclonal antibody (Product # 459250)

PGK1 Antibody — Figure 1--figure supplement 1. Effect of fluorophore tagging of ESCRT-III subunits and Vps4 on the endosomal traffic of cargo to the vacuole. ( a-c ) SDS-PAGE and western blot analysis using the indicated antibodies from total cell lysates ( a ) or equal volumes of subcellular fractions ( b, c ). Input (I), membrane fraction (M) and cytosol (C) samples for the various yeast strains are shown. Pgk1 was used as loading control. ( d ) Live cell epifluorescence and phase contrast microscopy of yeast cells expressing Vps4-mCherry (red) with Mup1-eGFP (green), Vps4-mNeonGreen (green) with Mup1-mScarlet (red) or Snf7-eGPF with or without endogenous Snf7 and Mup1-mCherry (red) 60 min after the addition of Methionine. Vacuole (V) and class E compartment (E) are indicated. Size bar = 5 um. ( e ) Effect of canavanine in the growth of yeast cells. Canavanine sensitivity assays were carried with different strains as indicated in the presence of 1 ug /ml L-canavanine. ( f ) Live cell epifluorescence and phase contrast microscopy of yeast cells carrying different ESCRT deletion mutants and expressing untagged Snf7 and Snf7-eGFP, Vps24-eGFP or Vps4-eGFP together with cargo (Mup1-mRuby2 or mCherry-CPS). These experiments constitute a positive control showing that the endosomal localization of the fluorophore-tagged ESCRT-III components or Vps4 depends on an active functional ESCRT pathway. Plasma membrane (PM), Vacuole (V) and class E compartment (E) are indicated. Size bar = 5 um.

PGK1 Antibody — Figure 3. Pulse-chase analysis of peroxisomal Pex15 turnover by Msp1. ( A ) Experimental timeline of a pulse-chase analysis similar to the one described in Figure 1A but with 10 ug/ml DOX. This experiment was performed twice with similar results. ( B ) Representative confocal micrographs from the experiment described in part A. Each image represents a maximum intensity projection of a Z-stack. Red cell outlines originate from a single bright-field image acquired at the center of the Z-stack. Scale bar, 5 mum. Contrast-enhanced YFP-Pex15 fluorescence is shown for the last timepoint +beta-estradiol to permit visualization of dim signals. ( C ) Quantitation of peroxisomal YFP-Pex15 fluorescence from the experiment described in part A . YFP-Pex15 fluorescence density corresponds to the total YFP-Pex15 signal at each computationally-defined peroxisome (marked by mCherry-PTS1) divided by the peroxisomal pixel volume (see Materials and methods for more details). Shown are violin plots of the resulting YFP-Pex15 density distributions. Solid lines represent cells with Msp1 expression driven by the beta-estradiol-inducible ZD promoter. Dashed lines represent cells with Msp1 produced from the endogenous MSP1 promoter. These data represent analysis of 270 mock-treated and 304 beta-estradiol-treated pMSP1-MSP1 cells and 219 mock-treated and 319 beta-estradiol-treated pZD-MSP1 cells followed throughout the time course as well as progeny from cell divisions during the experiment. The 515 nm

PGK1 Antibody — Figure 7. Rio1 responds to nutritional deprivation and crosstalks with Gcn4 at the transcriptional level. ( A ) The Rio1 transcriptome is enriched in transcripts of genes that are part of various stress-response systems. The main pie shows the percentage of the 818 Rio1-modulated genes that belong to specific stress transcriptomes (represented by the peripheral pies, which are size-weighted for the number of genes they contain (listed in Supplementary Table S10 ). The slices indicate the % of overlap at the gene level between the Rio1 and stress-response transcriptomes. P -values quantitating the significance of these overlaps (determined with Fischer's exact test) are listed in the text. ( B ) Expression analysis of 6 Rio1-regulated reporter genes in yeast growing in complete synthetic medium (+amino acids [AA] +2% glucose) or in synthetic medium lacking AA or glucose. Blue: gene whose transcript levels decrease due to Rio1 activity. Red: gene whose transcript levels increase due to Rio1 activity. White bars: Rio1-containing cells, black bars: Rio1-depleted cells. Transcript concentrations were measured by TaqMan-based RT-qPCR analysis and normalized to those of ACT1 (encoding actin, ACT1 mRNA levels are plotted in the upper right graph). Numbers were then correlated to those calculated for the mock-treated cells grown in complete synthetic medium. Error bars = standard errors. The RT-qPCR data are listed in Supplementary Table S11 . The levels of confidence ( P -v

PGK1 Antibody — Published figure using PGK1 monoclonal antibody (Product # 459250)

PGK1 Antibody — Figure 1 Identification of SidJ substitution mutants unable to suppress SdeA yeast toxicity. (A ) Distribution of substitution mutations that abolished the ability of SidJ to suppress the yeast toxicity of SdeA. Note the clustering of mutations around the 540th residue of SidJ. ( B) Yeast strain expressing chromosomally integrated SdeA controlled by a galactose-inducible promoter was transformed with plasmids carrying WT or mutated SidJ controlled by the alcohol dehydrogenase (ADH) promoter. Serially diluted yeast cells were spotted onto glucose or galactose medium. Images were acquired 3 days after incubation at 30 degC. Lower panel showed the expression of SdeA and SidJ, yeast cells grown in medium supplemented with glucose (1) were induced with galactose (2) for 8 h, and the total proteins separated by SDS-PAGE were detected by immunoblotting with antibodies for SdeA or SidJ, respectively. The 3-phosphoglycerate kinase (PGK) was probed as a loading control. (C) 293T cells were transfected with the plasmid that directs the expression of the secreted embryonic alkaline phosphatase (SEAP), GFP-SdeA and SidJ or its mutants for 24 h. The activity of SEAP in culture supernatant or the cells was measured to calculate the SEAP index. GFP and GFP-SdeA E/A that expressed the SdeA mutant defective in E 860 and E 862 , two residues critical for the mono-ADP-ribosyltransferase activity important for the activation of ubiquitin by ADP-ribosylation were used as controls. Lower panel show

PGK1 Antibody — FIGURE 2: Proteotoxic phenotypes of the hac1Deltalpl1Delta mutant. (A) Growth of wild-type, lpl1Delta , hac1Delta , and hac1Deltalpl1Delta strains in the presence of canavanine (1.5 ug/ml) or at elevated temperature, as indicated. Cells were spotted in threefold serial dilutions and cultured for 2-4 d. (B) Growth of wild-type, lpl1Delta , hac1Delta , and hac1Deltalpl1Delta strains expressing an empty vector and hac1Deltalpl1Delta expressing LPL1 in the presence or absence of canavanine (1.5 ug/ml), as indicated. Cells were spotted in threefold serial dilutions and cultured for 2-4 d at 30degC. (C) Splicing of HAC1 , as determined by RT-PCR, in wild-type and lpl1Delta strains. A wild-type strain treated with tunicamycin (5 ug/ml), an inducer of the unfolded protein response, serves as a positive control. ACT1 (bottom) serves as a control. (D) Kar2 protein levels in whole-cell extracts of wild-type and lpl1Delta strains, as determined by SDS-PAGE followed by immunoblot with anti-Kar2 antibody (top) or anti-Pgk1 antibody (bottom; loading control). (E) Kar2 protein levels in wild-type, lpl1Delta , hac1Delta , and hac1Deltalpl1Delta strains. Exponential-phase cells cultured in synthetic medium were treated at 37degC for 1 h. Whole-cell extracts were prepared and analyzed by SDS-PAGE followed by immunoblot with anti-Kar2 antibody (top) or anti-Pgk1 antibody (bottom; loading control).

PGK1 Antibody — FIGURE 3: Protein degradation defects in the hac1Deltalpl1Delta mutant. (A) Levels of ubiquitin conjugates in whole-cell extracts of wild-type, lpl1Delta , hac1Delta , and hac1Deltalpl1Delta strains, as determined by SDS-PAGE followed by immunoblot with anti-ubiquitin antibody (top) or anti-Pgk1 antibody (bottom; loading control). Experiment was performed at 37degC. (B) Cycloheximide chase analysis of CPY* turnover in wild-type, lpl1Delta , hac1Delta , and hac1Deltalpl1Delta strains, as determined by SDS-PAGE followed by immunoblot with anti-HA antibody (top) or anti-actin antibody (bottom; loading control). Experiment was performed at 37degC. See Supplemental Figure S1 for quantitation. (C) Cycloheximide chase analysis of turnover of the cytoplasmic proteasome substrate Delta2-GFP, as determined by SDS-PAGE followed by immunoblot with anti-HA antibody (top) or anti-Pgk1 antibody (bottom; loading control). Experiment was performed at 37degC. (D) Growth of wild-type, lpl1Delta , hac1Delta , and hac1Deltalpl1Delta strains expressing galactose-inducible wild-type STE6 or the misfolded mutant ste6-Q1249X. Cells were spotted in threefold serial dilutions and cultured for 4 d at 35degC. (E) Levels of phosphorylated eIF2alpha (Ser-51) in whole-cell extracts of wild-type, lpl1Delta , hac1Delta , and hac1Deltalpl1Delta strains treated with sodium arsenite (0.2 mM), as determined by SDS-PAGE followed by immunoblot with anti-phospho-eIF2alpha antibody (top) or anti-Pgk1 antibody (bottom

PGK1 Antibody — Figure 3. A functional Rpb4/7 heterodimer is a requisite for Sub1 recruitment to gene promoters. ( A ) Chromatin immunoprecipitation (ChIP) analyses were performed using wt and rpb4 Delta strains. Left panel: Rpb1 binding to the promoter (P) of four constitutively expressed genes, ADH1, PGK1, PMA1 and YEF3 was examined by qPCR. Results were quantified (see Materials and Methods), and relative Rpb1 binding in rpb4 Delta cells is plotted relative to that in wt cells (set equal to 100). The data plotted correspond to mean values from at least three independent experiments, and the error bars represent standard deviations. Middle and Right panels show relative Rpb3 and Sub1-HA binding, respectively, plotted as for Rpb1. ( B ) Plots of Sub1-HA/Rpb1, Sub1-HA /Rpb3 ratios in wild-type ( wt ) and rpb4 Delta using data from Figure 1A . ( C ) Whole cell extracts (WCE) were prepared from wild-type ( wt ) and rpb4 Delta strains expressing Sub1-HA and analyzed by western blotting using the following antibodies: anti-HA, anti-Rpb1 (8W16G), anti-Rpb3, anti-Rpb4 (2Y14) and anti-Pgk1 as a control for total protein. ( D ) Whole cell extracts (WCE) were prepared from wild-type ( wt ) and rpb4 Delta strains expressing Rpb7-HA and analyzed by Western blotting using anti-HA, anti-Rpb4 and anti-Pgk1. ( E ) Left panel: ChIP analysis in wt and rpb1-L1397S cells as in (A) to analyze Sub1-TAP occupancy at the promoter (P) of three constitutively expressed genes ( PMA1, ADH1 and PGK1 ) and to the promot

PGK1 Antibody — Figure 1. Sub1 interacts with the RNAPII through the Rpb4/7 heterodimer. ( A ) Sub1 tandem affinity purification from Sub1-TAP and Sub1-TAP Rpb7-HA whole cell extracts (WCE). Input and the purified proteins were precipitated with trichloroacetic acid and analyzed by western blotting using anti-TAP and anti-HA. Anti-Pgk1 was used as a loading control. ( B ) Co-IP performed using WCEs from Sub1-TAP (Rpb7-HA and Rpb7-HA/Rpb4-HA) with IgG Sepharose. Input and IPs were analyzed by western blotting with antibodies to the indicated proteins.

PGK1 Antibody — Figure 7. A full length Rpb1-CTD is required for efficient Sub1 association to gene promoters. ( A ) Genetic interaction between sub1 Delta and rpb1-CTD11 . The sub1Delta deletion increases the slow growth phenotype of rpb1-CTD11 strain at 16, 28 and 37degC. Serial dilutions (1:10) of wt and mutant strains were spotted on rich medium and grown for 2-3 days at the indicated temperatures. ( B ) Chromatin immunoprecipitation (ChIP) analyses were performed using wt and rpb1-CTD11 strains. Left panel: Rpb3, Rpb4 and Sub1-TAP binding to the promoter (P) of three constitutively expressed genes, PMA1, ADH1 and PGK1 , and to the promoter of the IMD2 inducible gene, was examined by qPCR. Results were quantitated (see Material and Methods), and relative Rpb3, Rpb4 and Sub1-TAP binding in rpb1-CTD11 cells is plotted relative to that in wt cells (set equal to 1). Right panel: WCE were prepared from wild-type ( wt ) and rpb1-CTD11 strains expressing Sub1-TAP and analyzed by Western blotting using the indicated antibodies. ( C ) Same as in (B) for wt and rpb1-CTD20 strains. ( D ) Left panel: Co-IP performed on WCEs from Sub1-TAP cells ( wt and rpb1-CTD11 ) using an anti-Rpb3 antibody. As a control, the same amount of cell extract was incubated with Protein A Sepharose alone. Inputs and IPs were analyzed by Western blotting with antibodies to the indicated proteins. Right panel: Values from the quantification of Sub1-TAP and Rpb3 immunoreactive signals from three experiments were calcuated a

PGK1 Antibody — Figure 3 In vivo characterization of Ssb1Delta601-13 shows complementation of pleiotropic ssb1,2 Delta phenotypes. ( a ) Schematic overview of Ssb domains and constructs used. Ssb1Delta601-13 lacks 13 C-terminal residues. ( b ) Growth analysis of Ssb-GFP constructs. Wild type (wt) or ssb1,2 Delta cells transformed with empty vector or Ssb-GFP versions were adjusted to OD 600 =0.4 and spotted in fivefold serial dilutions onto a YPD-plate, which was incubated at 30 degC for two days. ( c ) Cellular distribution of yEGFP-Ssb1 and yEGFP-Ssb1Delta601-13 analysed by confocal fluorescence microscopy. Transformed cells were grown to early exponential phase and applied onto agarose slices. Differential interference contrast (DIC) with scale bar=5 mum; DAPI was used for nuclei staining, overlay merges DAPI and GFP. ( d ) For polysome profiling wt cells transformed with empty vector (grey) or ssb1,2 Delta cells transformed with either empty vector (blue) or Ssb1 constructs (green, orange) were grown to early exponential phase. Lysates were adjusted and 18 A 260 units on top of a 15-45% (w/v) sucrose gradient were ultracentrifuged followed by gradient fractionation from top to bottom and OD 254 monitoring (top). Fractions were analysed via immunoblotting (bottom). Wt profiles at the background serve as control. ( e ) Protein expression of wt or ssb1,2 Delta cells transformed with either empty vector or Ssb1 constructs. Cells were grown to early exponential phase, lysed and protein levels

PGK1 Antibody — Fig. 2. GFP-Atg8 translocation into the vacuole via autophagy was suppressed in the Deltaole1 mutant. (A) Wild-type (BY4741), Delta atg1 (EUROSCARF), or Delta ole1 cells (BY4741-ole1) harboring pRS316/GFP-Atg8 were grown in SC-Ura+OA, shifted to SC-URA-OA for 2 h (+N-OA), and incubated further in SD-N without OA for 3 h (-N-OA). Cells were observed under a fluorescence microscope. Scale bars: 10 mum. (B) Wild-type (BY4741) or Delta ole1 cells (BY4741- ole 1) were grown in YPD+OA, shifted to YPD without OA (+N-OA) for 2 h, and further incubated in SD-N medium for 1 h (-N-OA). Cell lysates were processed for immunoblot analysis to detect Atg13, Atg8, and Pgk1 (used as an endogenous control).

PGK1 Antibody — Figure 5. Mutation of the predicted binding sites for Atg19 in Atg5 impairs the recruitment of the Atg12~Atg5-Atg16 complex to cargo mimetic beads and Cvt pathway function but does not affect bulk autophagy. ( A ) Coomassie stained gels showing the input amounts of the Atg12~Atg5-Atg16-mCherry complex (upper gel) and of the GST-prApe1(1-41) + Atg19 or GST proteins on the beads (lower gel) used for the experiment shown in ( B ). ( B ) GST-prApe1(1-41) + Atg19 or GST coated glutathione beads imaged in the presence of Atg12~Atg5-Atg16-mCherry complex (wild type, K57E, N84E or K57E,N84E). ( C ) Quantification of three independent experiments of the relative mCherry signal intensity measured at the beads. One experiment used for the quantification is shown in ( B ). The signal measured for the wild type Atg5~Atg12-Atg16-mCherry complex was set to 100%. Due to optical reasons very low signals at the beads resulted in values lower than the background and thus negative values. Error bars represent standard deviations. ( D ) Coomassie-stained gel showing the result of a liposome co-sedimentation assay using wild type Atg12~Atg5-Atg16-mCherry and the indicated point mutants thereof. Liposome binding allows the protein to be pelleted (P). The unbound protein remains in the supernatant (S). ( E ) Quantification of in vitro Atg8 conjugation assays using the indicated mutants of Atg12~Atg5. The amount of conjugated Atg8 and un-conjugated Atg8 was measured as the band intensity signal on a

PGK1 Antibody — Figure 6. CK2 phosphorylation sites are critical for Spt6 stability and chromatin functions. ( A ) CK2 mutant is associated with a decrease of Spt6 cellular level. Flag-Spt6 and Iws1-Myc proteins were analyzed by western blots on total extracts from wild type (WT) or ck2 ts heat-shocked cells using antibodies against Flag and Myc epitopes. ( B ) Mutation in CK2-dependent phosphosites leads to a decrease in Spt6 protein level. Flag-Spt6 and Iws1-Myc proteins were analyzed by western blots on chromatin extracts of cells expressing Flag-Spt6 or Flag-Spt6-7SA. ( C ) CK2-dependent phosphorylation of Spt6 is required for its stability. Flag-Spt6, Flag-Spt6-7SA (non-phosphrylatable) and Flag-Spt6-7SD (phosphomimic) proteins were analyzed by western blots on total extracts from heat-shocked cells treated for the time indicated with cycloheximide (CHX). Pgk1 was used as a loading control. Mutation of Spt6 phosphosites to alanine reduces its half-life while the change toward aspartic acid residues stabilizes this protein. ( D ) Spt6 phosphomimic mutant spt6-7SD suppresses the increased H3K56ac deposition observed in ck2 ts mutant. ChIP assays assessing the H3K56 acetylation level in WT and ck2 ts expressing Spt6 or Spt6-7SD. Cells were G1-arrested and subsequently grown for 1 h at 30 or 37degC. The values shown represent the average and standard errors of three independent experiments measuring H3K56ac levels (IP/Input) relative to histone H3 occupancy (IP/Input). Coding regi

PGK1 Antibody — Figure 5--figure supplement 2. Effect of the disruption of the Atg16 - Atg21 interaction on prApe1 processing. prApe1 processing assay in atg16Delta yeast strain expressing transiently transformed Atg16 D101A or E102A or D101A,E102A-double mutant proteins and Atg5-TAP or Atg5 K57E,N84E-TAP stably integrated in the genome (complete blot from Figure 5H upper blot). The Atg5 K57E,N84E-TAP in combination with Atg16 wild type shows reduced prApe1 processing. This mutant, in combination with Atg16 D101A,E102A and D101A,E102A-double mutation, shows fully arrested prApe1 processing. Atg5 expression levels were checked using anti-TAP-antibody; Pgk1 served as a loading control. DOI: http://dx.doi.org/

PGK1 Antibody — Figure 5 The increase of Ate1 triggers cell death in yeast in a manner that is dependent on its arginylation activity. ( a ) The scheme in the top panel shows the domain structure of plasmid pGAL1: ATE1 , in which the coding sequence of recombinant protein is preceded by the inducible GAL1 promoter. The picture in the bottom panel shows the growth of ate1 Delta yeast cells carrying either the empty expression vector or pGAL1: ATE1 by a serial dilution growth assay on either plate containing glucose (suppressing) or galactose (inducing). ( b ) Graph showing the viabilities of ate1 Delta yeast cells carrying either the empty expression vector or pGAL1: ATE1 in different time points following the initiation of galactose-induced expression, as measured by the numbers of colony-forming cells per OD unit (CFU) that were normalized to starting data point time 0, for Ate1 or vector control separately. Error bar represents S.E.M. ( n 3). ( c ) The top scheme shows the domain structure of a recombinant Ate1 fused with a linker and a C-terminal GFP, driven by the pGAL1 promoter, termed and apos; pGAL : Ate1-GFP and apos;. Immunoblot analysis of the steady-state levels of wild-type and C20,23S mutant Ate1 after 9-h culture in the presence of non-inducing (glucose) or inducing (galactose) carbon sources. PGK was used as loading controls. Anti-GFP was used to probe the steady-state levels of the recombinant and apos; pGAL : Ate1-GFP and apos; (WT or mutant). ( d ) Left panel showing the procedure of usi

PGK1 Antibody — Figure 4 The levels of Ate1 protein and global arginylation activity are upregulated during stress. ( a ) A scheme illustrating how DD- beta 15-GFP is used as the reporter of arginylation activity. The fusion protein containing a stretch of 15 amino acids starting with two aspartic acids (D) derived from the N terminus of mammalian beta -actin, a known substrate of arginylation. 31 This peptide is fused with an N-terminal ubiquitin, which is cleaved co-translationally by endogenous de-ubiquitylation enzymes in eukaryotic system and leaves the aspartic acids as the new N terminus. The arginylation state of this reporter can be probed with an anti-RDD antibody, which only reacts with the arginylated form of the reporter protein. A C-terminal GFP tag is used to facilitate the detection of steady-state level of the reporter protein by immunoblotting with anti-GFP antibody. ( b ) The arginylation level of DD- beta 15-GFP expressed in either WT or ate1 Delta yeast was examined with anti-RDD antibody, which only shows a visible signal in the WT cells. An antibody for GFP was used to probe the total protein level of the DD- beta 15-GFP. PGK was used as a loading control for total yeast cellular proteins. ( c ) Illustrative scheme (left panel) and representative immunoblots (right panel) showing the arginylation activity in cell lysates prepared from yeast exposed to 1 M NaCl stress for increasing times. The lysates were then mixed with the recombinant protein DD- beta 15-GFP prepared

PGK1 Antibody — Figure 6. Roles of the Atg38 C-terminal domain in dimerization, interaction with complex I and cellular fractionation. (A) All Atg38 C-terminal truncations cause overexpression of the Atg38 constructs. Total cell lysates from growing and nitrogen starvation conditions were subjected to western blotting. The same C-terminal deletion fragments as used in Fig. 5 were C-terminally tagged with 3X FLAG on a CEN plasmid (pRS416). The plasmids were transformed into atg38 Delta cells, and grown to mid-log phase (OD 600 = 0.8-1.0; Growing), then shifted to SD(-N) for 4 h. Total cell lysates were prepared as described in Materials and Methods. Asterisks indicate proteins of interest. Pgk1 expression was used as a loading control. (B) Atg38 C-terminal truncations cause dissociation of the proteins from the P15 pellet. Subcellular fractionation of the same proteins as in (A). Cells were grown to mid-log phase (OD 600 = 0.8-1.0; Growing), then shifted to SD(-N) medium and the total cell lysates (T), and supernatant (S15) and pellet (P15) fractions were analyzed. Pho8 was used as a membrane fraction (P15) control. (C) The C-terminal, crystallized region is important for homodimerization of Atg38. Immunoprecipitation between Atg38-ZZ and the same Atg38 truncations used above during nitrogen starvation. Yeast strain with chromosomally ZZ-tagged ATG38 was transformed with the same plasmids as (A) and (B). Cells were grown to mid-log phase, then shifted to SD(-N) for 4 h. The truncated proteins

PGK1 Antibody — Figure 5 MAPK phosphorylation dynamics and growth curves reveal the role of the receptor-RGS interaction in controlling both mating pathway activation and deactivation. ( a - f ) Levels of phosphorylated Fus3 after pheromone wash-off for different Ste2 variants. Following mating pathway activation with 3 muM pheromone cells, were washed in pheromone-free medium, incubated for the indicated time and lysed. Lysates were used for Western Blotting. Phosphorylated Fus3 levels were normalized to PGK levels and plotted. Green bar graphs indicate Scer alpha-factor, red indicated Klac alpha-factor. ( g - h ) Growth curves of cells expressing different Ste2 or Sst2 variants. Following mating pathway activation with 3 muM pheromone, cells were washed in pheromone-free medium, incubated for the indicated time and culture density was measured by flow cytometry. Values were normalized to the initial cell concentration to yield a growth ratio. All error bars represent the s.e.m.

PGK1 Antibody — Published figure using PGK1 monoclonal antibody (Product # 459250)

PGK1 Antibody — Published figure using PGK1 monoclonal antibody (Product # 459250)

PGK1 Antibody — Published figure using PGK1 monoclonal antibody (Product # 459250)

PGK1 Antibody — Published figure using PGK1 monoclonal antibody (Product # 459250)

PGK1 Antibody — Published figure using PGK1 monoclonal antibody (Product # 459250)

PGK1 Antibody — Figure 2. Uptake of beta-ala via the endogenous Put4 or the heterologous HcGap1 permease also promotes TORC1 activation. ( A ) w-t, gap1Delta , and gap1Delta put4Delta cells were grown on Gluc urea medium. [ 14 C]-beta-ala (0.1 or 2 mM) was added to the medium before measuring the incorporated radioactivity at various times. ( B ) Top. Cells as in A expressing HA-Npr1 from a plasmid were grown on Gluc urea medium. Cells were collected before and 4 and 10 min after addition of beta-ala (0.1 or 2 mM). Crude extracts were prepared and immunoblotted with anti-HA and anti-Pgk antibodies. Bottom. Same as in panel B except that gap1Delta cells were also collected 30 min after Rap treatment. ( C ) w-t and gap1Delta cells expressing or not HcGap1 from a plasmid were grown on Gluc Pro medium. [ 14 C]-beta-ala (0.5 or 2 mM) was added to the medium before measuring the incorporated radioactivity at various times. ( D ) Top. w-t and gap1Delta cells expressing HA-Npr1 and gap1Delta cells co-expressing HcGap1 and HA-Npr1 from plasmids were grown on Gluc Pro medium. Cells were collected before and 4 and 10 min after addition of beta-ala (0.5 or 2 mM). Crude extracts were prepared and immunoblotted with anti-HA and anti-Pgk antibodies. Bottom. Same as in top panel except that cells were also treated for 30 min with Rap before beta-ala addition.

PGK1 Antibody — Published figure using PGK1 monoclonal antibody (Product # 459250)

Product Details

Tested Applications

Dilution

Immunocytochemistry (ICC)

5-10 µg/mL

Immunofluorescence (IF)

5-10 µg/mL

Western Blot (WB)

1.0 µg/mL

Published Applications

Western Blot (WB)

See 108 publications below

Miscellaneous PubMed (MISC)

See 14 publications below

Product Specifications

Species Reactivity

Yeast

Published species

Human, Mouse, Yeast

Host / Isotype

Mouse / IgG1, kappa

Class

Monoclonal

Type

Antibody

Clone

22C5D8

Immunogen

Yeast pGK (3-Phosphoglyceric Phosphokinase), EC 2.7.2.3

Conjugate

Unconjugated

Form

Liquid

Concentration

1 mg/mL

Purification

Ammonium sulfate precipitation

Storage buffer

HEPES buffered saline

Contains

0.02% sodium azide

Storage conditions

4° C

RRID

AB_2532235

Target Information

The protein encoded by this gene is a glycolytic enzyme that catalyzes the conversion of 1,3-diphosphoglycerate to 3-phosphoglycerate. The encoded protein may also act as a cofactor for polymerase alpha. This gene lies on the X-chromosome, while a related pseudogene also has been found on the X-chromosome and another on chromosome 19.

For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.

Bioinformatics

Protein Aliases: cell migration-inducing gene 10 protein; MIG10; PGKA; Phosphoglycerate kinase; primer recognition protein 2

Gene Aliases: PGK1; YCR012W; YCR12W

UniProt ID: (Baker's yeast) P00560

Entrez Gene ID: (Baker's yeast) 850370

Molecular Function: carbohydrate kinase kinase transferase

gluconeogenesis glycolytic process phosphorylation

Process(es)

nucleotide binding phosphoglycerate kinase activity ATP binding kinase activity transferase activity

Suggested Secondary Antibodies

Anti-Mouse Secondary Antibodies (266)
Alexa Fluor® Anti-Mouse Secondary Antibodies (76)
HRP Anti-Mouse Secondary Antibodies (28)