Total lysates (15 µg protein) from untreated HeLa cells (lane 1) and HeLa cells treated with nocodazole (lane 2) were resolved by electrophoresis (4-12% Bis-Tris gel), transferred to nitrocellulose, and probed with 1:500 diluted (1 µg/mL) Purified anti-STAT1 Phospho (Ser727) Antibody, clone A15158B (upper) or 1:3000 diluted Purified anti-β-actin Antibody, clone Poly6221 (lower). Proteins were visualized by chemiluminescence detection using a 1:3000 diluted goat anti-mouse-IgG secondary antibody conjugated to HRP for the anti-STAT1 Phospho (Ser727) Antibody, and a donkey anti-rabbit IgG Antibody conjugated to HRP for anti-β-actin Antibody.
Total lysates (15 µg protein) from untreated HeLa cells (lane 1) and HeLa cells treated with nocodazole (lane 2) were resolved by electrophoresis (4-12% Bis-Tris gel), transferred to nitrocellulose, and probed with 1:500 diluted (1 µg/mL) Purified anti-STAT1 Phospho (Ser727) Antibody, clone A15158B (upper) or 1:3000 diluted Purified anti-β-actin Antibody, clone Poly6221 (lower). Proteins were visualized by chemiluminescence detection using a 1:3000 diluted goat anti-mouse-IgG secondary antibody conjugated to HRP for the anti-STAT1 Phospho (Ser727) Antibody, and a donkey anti-rabbit IgG Antibody conjugated to HRP for anti-β-actin Antibody.
Total cell lysates (15 µg total protein) from NIH/3T3 cells untreated (-) or treated (+) with UV radiation (20 mJ for 5 seconds) were resolved by 4-12% Bis-Tris gel electrophoresis, transferred to a PVDF membrane, and probed with 1.0 µg/mL (1:500 dilution) of purified anti-STAT1 Phospho (Ser727) antibody (clone A15158B) overnight at 4°C. Proteins were visualized by chemiluminescence detection using HRP goat anti-mouse IgG antibody (Cat. No. 405306) at a 1:3000 dilution. Purified anti-STAT1 antibody (clone 10C4B40) (Cat. No. 661002), was used as a pan STAT1 loading control. Direct-Blot™ HRP anti-GAPDH antibody (Cat. No. 607904) was used as a loading control at a 1:25000 dilution (lower). Lane M: Molecular weight marker.
HeLa cells were stimulated with (filled histogram) or without (open histogram) nocodozole for 24 hours, fixed with Fixation Buffer, permeabilized with True-Phos™ Perm Buffer, then intracellularly stained with purified anti-STAT1 Phospho (Ser727) antibody (clone A15158B), followed by anti- mouse IgG PE.
Human peripheral blood lymphocytes were stimulated with (filled histogram) or without (open histogram) Cell Activation Cocktail (without Brefeldin A) for 15 minutes, fixed with Fixation Buffer, permeabilized with True-Phos™ Perm Buffer, then intracellularly stained with purified anti-STAT1 Phospho (Ser727) antibody (clone A15158B), followed by anti- mouse IgG PE.
Two aliquots of HeLa cells in suspension, treated with (top) 200 ng/mL nocodazole for 24 hours or left un-treated (bottom) were adhered on poly-lysine pre-coated slides. Then they were fixed with 4% paraformaldehyde (PFA) for 15 minutes, permeabilized with 0.5% Triton X-100 for three minutes, and blocked with 5% FBS for 60 minutes. The cells were intracellularly stained with 2 µg/ml of purified anti-STAT1 Phospho (Ser727) antibody (clone A15158B) overnight at 4°C followed by Alexa Fluor® 594 (red) conjugated goat anti-mouse IgG for one hour at room temperature. Nuclei were counterstained with DAPI (blue). The image was captured with a 60X objective.
Chromatin Immunoprecipitations (ChIP) were performed with cross-linked chromatin samples from 4 X 106 of HeLa cells treated with Nocodazole with either A)1:50 dilution of Go-ChIP-Grade™ Purified anti-STAT1 Phospho (Ser727) Antibody (clone A15158B, Cat. No. 686413) or B) equal amount of Purified Mouse IgG1, κ Isotype Control Antibody (Clone MG1-45, Cat. No. 401401) by using Go-ChIP-Grade™ Protein G Enzymatic Kit (Cat. No. 699904). The enriched DNA was purified and quantified by real-time qPCR using primers targeting human IRF1 gene region or α-Satellite repeats. The amount of immunoprecipitated DNA in each sample is represented as signal relative to total amount of input chromatin.
STAT1, also known as signal transduction and activator of transcription 1, is a ubiquitously expressed cytoplasmic protein and is activated in response to cytokine signaling, including IFN-α, IFN-γ, EGF, PDGF, and IL-6. Upon activation, STAT1 is phosphorylated by receptor-associated kinases, translocates to the nucleus, and functions as a transcription factor. Two isoforms of STAT1, with apparent molecular weights of 88 and 91 kD, exist as a result of alternative RNA processing. STAT1 is involved in IFN-mediated immune responses, and STAT1-deficient mice are highly sensitive to bacterial and viral infections.
Each lot of this antibody is quality control tested by Western blotting. For Western blotting, the suggested use of this reagent is 0.5 - 2.5 µg/ml. For intracellular flow cytometry using our True-Phos™ Perm Buffer in Cell Suspensions Protocol, the suggested use of this reagent is ≤ 0.03 µg per million cells in 100 µl volume. For immunocytochemistry, a concentration range of 0.5 - 2.0 μg/ml is recommended. For ChIP applications, the suggested dilution is 1:50-1:100 by volume. It is recommended that the reagent be titrated for optimal performance for each application.
750 amino acids, predicted molecular weight of 87 kD; contains a SH2 domain responsible for homodimerization or heterodimerization.
Distribution
Translocates to the nucleus when phosphorylated.
Function
Phosphorylated in response to cytokine signaling by receptor-associated kinases; translocates to the nucleus to act as a transcription factor. Mediates responses to type I (IFN-α/β) and II interferon (IFN-γ), EGF, PDGF, and IL-6.
Interaction
Forms a homodimer or heterodimers with other family members. Interacts with FAK, MCM3, MCM5, TRADD, BRCA1, KIT, IL-27R, IL-2Rβ, IL-2Rγ, IFNαβR, and c-Src.
Biology Area
Cell Biology, Signal Transduction
Molecular Family
Nuclear Markers, Phospho-Proteins
Antigen References
1. Durbin JE, et al. 1996. Cell. 84:443.
2. Darnell JE Jr, et al. 1994. Science 264:1415.
3. Chen X, et al. 1998. Cell. 93:827.
4. Ramana CV, et al. 2000. Oncogene. 19:2619.
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