Western blot analysis of extracts from HeLa cells, untreated (-) or treated with IFN-alpha (100 ng/ml) for 5 min, using Stat1 (D1K9Y) Rabbit mAb #14994 (upper) and P-Stat1 (Tyr701) (58D6) Rabbit mAb #9167 (lower).
Western blot analysis of extracts from HeLa cells, untreated (-) or treated with IFN-alpha (100 ng/ml) for 5 min, using Stat3 (D3Z2G) Rabbit mAb #12640 (upper) and P-Stat3 (Tyr705) (D3A7) XP Rabbit mAb #9145 (lower).
Western blot analysis of extracts from HeLa cells, untreated (-) or treated with IFN-alpha (100 ng/ml) for 5 min, using Stat5 (D2O6Y) Rabbit mAb #94205 (upper) and P-Stat5 (Tyr694) (D47E7) Rabbit mAb #4322 (lower).
Boil for 3 minutes prior to use. Load 10 µl of phosphorylated and nonphosphorylated Stat1/2/3/5 Control Cell Extracts per lane.
Supplied in SDS Sample Buffer: 62.5 mM Tris-HCl (pH 6.8 at 25°C), 2% w/v SDS, 10% glycerol, 50 mM DTT, 0.01% w/v bromophenol blue or phenol red. Store at –20°C. For long term, store at –80°C.
Stat1/2/3/5 Control Cell Extracts (HeLa untreated): Total cell extracts from serum-starved HeLa cells serve as a negative control. Supplied in SDS Sample Buffer.
Stat1/2/3/5 Control Cell Extracts (HeLa + IFN-alpha): Total cell extracts from serum-starved HeLa cells treated with 100 ng/ml interferon-alpha for 5 minutes serve as a positive control. Supplied in SDS Sample Buffer.
Jaks (Janus Kinases) and Stats (Signal Transducers and Activators of Transcription) are utilized by receptors for a wide variety of ligands including cytokines, hormones, growth factors and neurotransmitters. Jaks, activated via autophosphorylation following ligand-induced receptor aggregation, phosphorylate tyrosine residues on associated receptors, Stat molecules and other downstream signaling proteins (1,2). The phosphorylation of Stat proteins at conserved tyrosine residues activates SH2-mediated dimerization followed rapidly by nuclear translocation. Stat dimers bind to IRE (interferon response element) and GAS (gamma interferon-activated sequence) DNA elements, resulting in the transcriptional regulation of downstream genes (1,2). The remarkable range and specificity of responses regulated by the Stats is determined in part by the tissue-specific expression of different cytokine receptors, Jaks and Stats (2,3), and by the combinatorial coupling of various Stat members to different receptors. Serine phosphorylation in the carboxy-terminal transcriptional activation domain has been shown to regulate the function of Stat1, -2, -3, -4 and -5 (1). Phosphorylation of Stat3 at Ser727 via MAPK or mTOR pathways is required for optimal transcriptional activation in response to growth factors and cytokines including IFN-gamma and CNTF (4,5). Jak/Stat pathways also play important roles in oncogenesis, tumor progression, angiogenesis, cell motility, immune responses and stem cell differentiation (6-11).
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