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Phospho-CREB (Ser129, Ser133) Polyclonal Antibody
Antibody target was verified by Relative expression to ensure the antibody binds to the antigen stated.
Catalog # 44-297G
(USD) 309.00, 10 blots
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Immunofluorescence analysis of CREB (pS129/pS133) 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 CREB (pS129/pS133) Rabbit polyclonal Antibody (Product # 44-297G) at 2 µg/mL in 1% BSA and incubated for 3 hours at room temperature and then labeled with Alexa Flour 488 Goat Anti-Rabbit IgG Secondary Antibody (Product # A-11008) 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 with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Alexa Fluor 594 Phalloidin (Product # A12381). Panel d is a merged image showing nuclear localization. Panel e shows no primary antibody control. The images were captured at 20X magnification.
Peptide Competition and Phosphatase Treatment. Extracts of NIH3T3 cells untreated (1) or treated with 50ng/mLPDGF for 15 minutes (2-6) were resolved by SDS-PAGE on a 10% Tris-glycine gel and transferred to PVDF. The membrane was blocked with a 5% BSA-TBST buffer for one hour at room temperature and either left untreated (1-5) or treated with lambda phosphatase (6), then incubated with the CREB [pSpS129/133] antibody (Product # 44-297G) for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1, 2, 6), the non-phosphopeptide corresponding to the phosphopeptide immunogen (3), a generic phosphoserine-containing peptide (4), or the phosphopeptide immunogen (5). After washing, the membrane was incubated with goat F(ab’)2 anti-rabbit IgG HRP conjugate (Product # ALI4404) and signals were detected using the Pierce SuperSignal™ method. The data show that only the phosphopeptide corresponding to CREB [pSpS129/133] blocks the antibody signal, demonstrating the specificity of the antibody. The data also show that phosphatase stripping eliminates the signal, further verifying that the antibody is phospho-specific. Note: Pretreatment of cells with PKA inhibitor (PKI) or GSK-3beta inhibitor (LiCl) diminished the signal, indicating the dual phosphoreactivity of this antibody (data not shown).
Western blot analysis of CREB (pS129/pS133) was performed by loading 20 µg of NIH/3T3 (lane1), NIH/3T3 treated for 10 minutes with 50 ng/ml of PDGF (lane2), NIH/3T3 treated for 10 minutes with 200 ng/ml of EGF (lane3), A549 (lane4), A549 treated for 10 minutes with 200 ng/ml of EGF (lane5), A431 (lane6), SK-N-SH (lane7), MCF7 (lane8) and MDA-MB-231 (lane9) cell lysate using Novex®NuPAGE® 12 % Bis-Tris gel (Product # NP0342BOX), XCell SureLock™ Electrophoresis System (Product # EI0002), Novex® Sharp Pre-Stained Protein Standard (LC5800), and iBlot® 2 Dry Blotting System (IB21001). Proteins were transferred to a nitrocellulose membrane and blocked with 5% skim milk for 1 hour at room temperature. CREB (pS129/pS133) was detected at ~ 35 and 43 kDa using CREB (pS129/pS133) Rabbit Polyclonal Antibody (Product # 44-297G) at 1:1000 dilution in 5% skim milk at 4°C overnight on a rocking platform. Goat Anti-Rabbit IgG - HRP Secondary Antibody (G21234) at 1:5000 dilution was used and chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (WP20005).
Whole cell extracts of human A549 cells (approximately 20,000 cells per lane) untreated (1), stimulated with EGF (2) or treated with pervanadate (3), were resolved by SDS-PAGE and transferred to PVDF. The membrane was blocked with a casein/Tween 20 buffer, then incubated with mouse anti-CREB/ATF1 (pS133) antibody (Product # 44297M) at 0.5 µg/mL for 1 hour at room temperature. After washing, the membrane was incubated with an anti-mouse HRP-conjugated secondary antibody and signals were detected using an ECL detection method (exposure time: 30 seconds).
ChIP- qPCR analysis of CREB (pSer133) was performed with 10µl of the CREB (pSer133) Rabbit polyclonal antibody (Product # 44-297G) on sheared chromatin from 2 million HeLa cells treated with 50ng/ml of TNFalpha for one hour using the MAGnify™ Chromatin Immunoprecipitation System (Product # 49-2024). Normal Rabbit IgG was used as a negative IP control. The purified DNA from each ChIP sample was analyzed by StepOnePlus™ Real-Time PCR System (Product # 4376600) with primers for the promoter of active YWHAZ, I-kB gene, used as positive control target, and the inactive GAPDH, used as negative control target. Data is presented as fold enrichment of the antibody signal versus the negative control IgG using the comparative CT method.
Antibody specificity was demonstrated by detection of enrichment of the target protein at specific gene loci. Chromatin Immunoprecipitation (ChIP) was performed using Phospho-CREB (Ser129, Ser133) Rabbit Polyclonal Antibody (Product # 44-297G) with relevant positive (YWHAZ, Ik-B) and negative (SAT2) sites.
Altered expression of proteins upon cell treatment demonstrates antibody specificity. Western blot analysis of Phospho-CREB (Ser129, Ser133) using Phospho-CREB (Ser129, Ser133) Rabbit Polyclonal Antibody (Product # 44-297G) shows induction of phosphorylation of CREB in NIH/3T3 and A549 cell lines upon treatment with PDGF and EGF respectively.
Altered expression of proteins upon cell treatment demonstrates antibody specificity. Western blot analysis of Phospho-CREB (Ser129, Ser133) using Phospho-CREB (Ser129, Ser133) Rabbit Polyclonal Antibody (Product # 44-297G) shows increased levels of Phospho-CREB (Ser129, Ser133) in A549 cell line upon EGF and Pervanadate treatment.
Altered expression of proteins upon cell treatment demonstrates antibody specificity. Western blot analysis of Phospho-CREB (Ser129, Ser133) using Phospho-CREB (Ser129, Ser133) Rabbit Polyclonal Antibody (Product # 44-297G) shows increased levels of Phospho-CREB (Ser129, Ser133) in NIH/3T3 cell line upon PDGF treatment.
ChIP assay (ChIP)
Western Blot (WB)
Host / Isotype
Phosphopeptide containing amino acid sequence RRP[pS]YRK, conjugated to KLH.
Antigen affinity chromatography
Dulbecco's PBS, pH 7.3, with 1mg/ml BSA, 50% glycerol
0.05% sodium azide
Cyclic AMP response element binding protein (CREB) is a basic/leucine zipper transcription factor that binds the cyclic AMP response element (CRE) and activates transcription in response to a variety of extracellular signals including neurotransmitters, hormones, membrane depolarization, and growth and neurotrophic factors. Activation of CREB is dependent upon the phosphorylation of serine 133. Phosphorylation occurs via p44/42 MAP kinase and p90RSK and also via p38 MAP kinase and MSK1. Although CREB will bind DNA independent of its phosphorylation state, only the phosphorylated form is competent as a transcription factor. CREB binding protein (CBP), a transcriptional coactivator that directly interacts with CREB, binds to CREB in the region of serine 133. CREB appears to play an important role in learning and memory.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
Protein Aliases: active transcription factor CREB; cAMP responsive element binding protein 1; cAMP-response element binding protein; cAMP-response element-binding protein-1; cAMP-responsive element-binding protein 1; CREB-1; CREB1; cyclic adenosine 3',5'-monophosphate response element-binding protein CREB; Cyclic AMP-responsive element-binding protein 1; transactivator protein
Gene Aliases: 2310001E10Rik; 3526402H21Rik; AV083133; CREB; CREB-1; CREB1
UniProt ID: (Human) P16220, (Mouse) Q01147
Entrez Gene ID: (Human) 1385, (Mouse) 12912
Suggested Secondary Antibodies
Material safety data sheets (MSDS)