Invitrogen

mTOR Monoclonal Antibody (215Q18)

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mTOR Antibody in Immunofluorescence (IF) — Immunofluorescent analysis of mTOR was done on 70% confluent log phase HeLa cells. The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.25% Triton™ X-100 for 10 minutes, and blocked with 5% BSA for 1 hour at room temperature. The cells were labeled with mTOR Mouse monoclonal Antibody (Product # AHO1232) at 2 µg/mL in 1% BSA and incubated for 3 hours at room temperature and then labeled with Alexa Fluor 488 Rabbit Anti-Mouse IgG Secondary Antibody (Product # A-11059) at a dilution of 1:400 for 30 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant DAPI (Product # S36938). F-actin (Panel c: red) was stained with Alexa Fluor 594 Phalloidin (Product # A12381). Panel d is a merged image showing cytoplasmic localization. Panel e shows no primary antibody control. The images were captured at 20X magnification.

mTOR Antibody in Immunohistochemistry (Paraffin) (IHC (P)) — Immunohistochemistry analysis of mTOR showing staining in the cytoplasm of paraffin-embedded human testis tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a mTOR Mouse Monoclonal Antibody (clone 215Q18) (Product # AHO1232) diluted in 3% BSA-PBS at a dilution of 1:50 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

mTOR Antibody in Western Blot (WB) — Western blot analysis of mTOR using a monoclonal antibody (Product # AHO1232).

mTOR Antibody — Figure 1 Knockdown of YAP1 and mTOR inhibited the progression of bladder cancer. (A and B) RT-PCR and western blot assays were performed to test the expression of YAP1 and mTOR in bladder cancer and normal bladder tissues ( *** P<0.001). (C) Immunohistochemistry was used to evaluate the expression patterns of the YAP1 and mTOR proteins in three matched pairs of bladder cancer and normal bladder tissues (magnification, x200). (D and E) Immunohistochemistry scores of mTOR and YAP1 staining in 20 paired bladder cancer and adjacent normal tissues. (F and G) RT-PCR was used to analyze the knockdown efficien-cies of si-YAP1 and si-mTOR ( * P<0.05, ** P<0.01). (H and I) HT-1376 and J82 cells were transfected with si-NC, si-YAP1, si-mTOR or si-YAP1 + si-mTOR for 0, 24, 48, 72 and 96 h; then, CCK-8 assay was performed to evaluate cell proliferation. (J) HT-1376 and J82 cells were transfected with si-NC, si-YAP1, si-mTOR or si-YAP1 + si-mTOR for 48 h; then, cells were collected for flow cytometry assay to detect cell apoptosis. (K) Western blotting was performed to detect the expression of apoptosis-related proteins, such as caspase 3/9 and cleaved-caspase3/9 after 48 h of cell transfection (H-K, vs. si-NC group, * P<0.05; vs. si-YAP1 group, # P<0.05; NC, negative control). YAP1, Yes-associated protein 1; mTOR, mammalian target of rapamycin; RT-PCR, reverse transcription-polymerase chain reaction.

mTOR Antibody — Figure 2 Crosstalk between the YAP1 and mTOR proteins. (A and B) HT-1376 and J82 cells were transfected with OE-mTOR and OE-NC; then cells were harvested and subjected to RT-PCR and western blot assays to determine the expression of mTOR at the mRNA and protein levels, respectively. (C-F) RT-PCR and western blot assays were performed to determine the mRNA and protein expression of YAP1 and TEAD after 48 h of HT-1376 and J82 cell transfection with OE-mTOR or OE-NC. (G) Immunofluorescence assay was performed to evaluate the effects of subcellular location of the YAP1 protein. (H) Statistical analysis of the fluorescence intensity of the YAP1 protein ( * P<0.05, ** P<0.01; NC, negative control). YAP1, Yes-associated protein 1; mTOR, mammalian target of rapamycin; RT-PCR, reverse transcription-polymerase chain reaction; TEAD, TEA domain transcription factor.

mTOR Antibody — Figure 4 Interaction between YAP1 protein and mTOR protein in HT-1376 and J82 cells. (A) Immunofluorescence assay was performed to evaluate the subcellular localization of the YAP1 and SKP2 proteins. (B) Duolink assay was performed to evaluate the subcellular localization of the YAP1 and mTOR proteins. (C and D) Western blotting was performed to assess the expression of flag and YAP1 after J82 and HT-1376 cells were transfected with flag-tag or YAP1-flag-tag vector ( * P<0.05). (E) Immunoprecipitation assay was used to assess the combination of YAP1 and mTOR proteins ['input' refers to total protein lysate and 'eluent' refers to the immune complex pulled down by YAP1 antibody or flag antibody; beads were used as a negative control (NC)]. YAP1, Yes-associated protein 1; mTOR, mammalian target of rapamycin; SKP2, S-phase kinase-associated protein 2.

Product Details

Applications

Tested Dilution

Publications

Immunocytochemistry (ICC)

1:100-1:500

View 2 publications 2 publications

Immunofluorescence (IF)

1:100-1:500

View 2 publications 2 publications

Immunohistochemistry (Paraffin) (IHC (P))

1:10-1:100

-

Western Blot (WB)

Assay Dependent

View 3 publications 3 publications

Immunohistochemistry (IHC)

-

View 1 publication 1 publication

Immunoprecipitation (IP)

-

View 1 publication 1 publication

Product Specifications

Species Reactivity

Human, Mouse, Rat

Published species

Mouse, Rat

Host / Isotype

Mouse / IgG2b, kappa

Class

Monoclonal

Type

Antibody

Clone

215Q18

Immunogen

Recombinant fragment of human mTOR expressed in E. coli.

Conjugate

Unconjugated

Form

Liquid

Purification

Affinity chromatography

Storage buffer

PBS, pH 7.2, with 1% BSA

Contains

0.1% sodium azide

Storage conditions

-20°C

RRID

AB_2536329

Target Information

Mammalian target of rapamycin (mTOR) is a serine/threonine kinase that plays a key role in cell growth, cell proliferation, and protein synthesis. mTOR mediates phosphoinositide 3-kinase and Akt/PKB signaling, resulting in phosphorylation of 4EBP1, and initiation of mRNA translation. A second pathway involves regulation of ribosomal S6 kinase, which affects ribosome biogenesis and translation elongation. Mutations affecting the gene results in Smith-Kingsmore syndrome.

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

Bioinformatics

Protein Aliases: angiopoietin-like factor CDT6; FK506 binding protein 12-rapamycin associated protein 1; FK506 binding protein 12-rapamycin associated protein 2; FK506-binding protein 12-rapamycin complex-associated protein 1; FKBP-rapamycin associated protein; FKBP-rapamycin associated protein (FRAP); FKBP-rapamycin-associated protein FRAP; FKBP12-rapamycin complex-associated protein; FKBP12-rapamycin complex-associated protein 1; m-TOR; Mammalian target of rapamycin; Mechanistic target of rapamycin; mechanistic target of rapamycin (serine/threonine kinase); mTOR; mTORC1; Rapamycin and FKBP12 target 1; rapamycin and FKBP12 target-1 protein; rapamycin associated protein FRAP2; Rapamycin target protein 1; RAPT1; Serine/threonine-protein kinase mTOR

Gene Aliases: 2610315D21Rik; AI327068; flat; FRAP; FRAP1; FRAP2; MTOR; RAFT1; RAPT1; SKS

UniProt ID: (Human) P42345, (Mouse) Q9JLN9, (Rat) P42346

Entrez Gene ID: (Human) 2475, (Mouse) 56717, (Rat) 56718

Molecular Function: kinase non-receptor serine/threonine protein kinase nucleic acid binding nucleotide kinase protein kinase transferase

negative regulation of protein phosphorylation positive regulation of protein phosphorylation positive regulation of endothelial cell proliferation heart morphogenesis heart valve morphogenesis regulation of glycogen biosynthetic process energy reserve metabolic process 'de novo' pyrimidine nucleobase biosynthetic process DNA repair protein phosphorylation response to stress cell cycle arrest signal transduction germ cell development brain development cell aging response to nutrient long-term memory visual learning post-embryonic development negative regulation of autophagy positive regulation of lamellipodium assembly positive regulation of gene expression positive regulation of myotube differentiation positive regulation of neuron maturation negative regulation of muscle atrophy cell growth macroautophagy negative regulation of macroautophagy phosphorylation peptidyl-serine phosphorylation peptidyl-threonine phosphorylation spinal cord development protein catabolic process positive regulation of actin filament polymerization T cell costimulation negative regulation of protein ubiquitination ruffle organization regulation of myelination cellular response to nutrient levels TOR signaling regulation of fatty acid beta-oxidation regulation of response to food response to insulin regulation of actin cytoskeleton organization social behavior multicellular organism growth growth wound healing response to cocaine regulation of GTPase activity response to amino acid response to morphine regulation of carbohydrate utilization positive regulation of nitric oxide biosynthetic process regulation of osteoclast differentiation positive regulation of translation negative regulation of cell size regulation of protein kinase activity positive regulation of transcription from RNA polymerase III promoter protein autophosphorylation positive regulation of lipid biosynthetic process phosphatidylinositol-mediated signaling mRNA stabilization positive regulation of smooth muscle cell proliferation positive regulation of oligodendrocyte differentiation positive regulation of peptidyl-tyrosine phosphorylation voluntary musculoskeletal movement positive regulation of stress fiber assembly negative regulation of NFAT protein import into nucleus positive regulation of protein kinase B signaling cardiac muscle cell development cardiac muscle contraction maternal process involved in female pregnancy positive regulation of glial cell proliferation positive regulation of dendritic spine development positive regulation of cell growth involved in cardiac muscle cell development cellular response to hypoxia regulation of brown fat cell differentiation regulation of membrane permeability regulation of cellular response to heat positive regulation of neuron death positive regulation of transcription of nuclear large rRNA transcript from RNA polymerase I promoter positive regulation of eating behavior positive regulation of cholangiocyte proliferation positive regulation of sensory perception of pain negative regulation of cholangiocyte apoptotic process positive regulation of granulosa cell proliferation positive regulation of skeletal muscle hypertrophy negative regulation of iodide transmembrane transport double-strand break repair via homologous recombination regulation of carbohydrate metabolic process cell projection organization

Process(es)

RNA polymerase III type 1 promoter DNA binding RNA polymerase III type 2 promoter DNA binding RNA polymerase III type 3 promoter DNA binding TFIIIC-class transcription factor binding protein kinase activity protein serine/threonine kinase activity protein binding ATP binding kinase activity protein kinase binding protein domain specific binding ribosome binding phosphoprotein binding nucleotide binding drug binding transferase activity transferase activity, transferring phosphorus-containing groups phosphotransferase activity, alcohol group as acceptor protein dimerization activity