|Detection of Human/Mouse STAT5a/b by Western Blot. Western blot shows lysates of K562 human chronic myelogenous leukemia cell line and M1 mouse myeloid leukemia cell line. PVDF membrane was probed with 1 µg/mL of Rabbit Anti-Human/Mouse STAT5a/b Pan Specific Antigen Affinity-purified Polyclonal Antibody (Catalog # AF2168) followed by HRP-conjugated Anti-Rabbit IgG Secondary Antibody (Catalog # HAF008). Specific bands were detected for STAT5a at approximately 94 kDa and STAT5b at approximately 92 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.|
Chromatin Immunoprecipitation (ChIP)
|Detection of STAT5a/b-regulated Genes by Chromatin Immunoprecipitation. BaF3 mouse pro-B cell line treated with 10 ng/mL Recombinant Mouse IL‑3 (Catalog # 403-ML) for 1 hour was fixed using formaldehyde, resuspended in lysis buffer, and sonicated to shear chromatin. STAT5a/b/DNA complexes were immunoprecipitated using 5 μg Rabbit Anti-Human/Mouse STAT5a/b Pan Specific Antigen Affinity-purified Polyclonal Antibody (Catalog # AF2168) or control antibody (Catalog # AB-105-C) for 15 minutes in an ultrasonic bath, followed by Biotinylated Anti-Rabbit IgG Secondary Antibody (Catalog # BAF008). Immunocomplexes were captured using 50 μL of MagCellect Streptavidin Ferrofluid (Catalog # MAG999) and DNA was purified using chelating resin solution. The bcl-x promoter was detected by standard PCR.|
|Detection of Human STAT5a/b by Simple WesternTM. Simple Western lane view shows lysates of HeLa human cervical epithelial carcinoma cell line and K562 human chronic myelogenous leukemia cell line, loaded at 0.2 mg/mL. A specific band was detected for STAT5a/b at approximately 91 kDa (as indicated) using 20 µg/mL of Rabbit Anti-Human/Mouse STAT5a/b Pan Specific Antigen Affinity-purified Polyclonal Antibody (Catalog # AF2168). This experiment was conducted under reducing conditions and using the 12-230 kDa separation system.|
Signal transduction and activator of transcription 5 (STAT5) is a member of the Jak/STAT signal transduction pathway and is activated by a variety of cytokines (IL22, IL6, IFN-α). STAT5 has two isoforms (A and B) that share 93% amino acid identity and bind the DNA consensus site TTCN3GAA. STAT5 mediates cytokine signaling by acting as a signal transducer in the cytoplasm and, upon phosphorylation, translocates to the nucleus and activates transcription of specific genes. STAT5 is involved in a wide array of biological processes ranging from regulating apoptosis to adult mammary gland proliferation, differentiation and survival.
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