P y694 699 stat5

Cells were suspended in Laemmli’s 2× buffer (Bio-Rad, Hercules, CA, USA), separated on SDS/PAGE and blotted onto nitrocellulose membrane. Blots were incubated with the following antibodies (Abs): P-Y^694/699-STAT5, Actin (Cell Signaling Technology, Danvers, MA, USA), STAT5 (BD Transduction Laboratories, Franklin Lakes, NJ, USA), STAT5A and STAT5B (Zymed/ThermoFisher Scientific, Waltham, MA, USA). Membranes were developed with the ECL chemiluminescence detection system (GE Healthcare, Little Chalfont Buckinghamshire, UK) using specific peroxidase (HRP) conjugated to rabbit or mouse IgG antibodies (Cell Signaling Technology).

NP40 cell lysates (1% NP40, 10% glycerol, 0.05 M Tris pH 7.5, 0.15 M NaCl, 1 mM PMSF, protease and phosphatase Inhibitor cocktails) (Roche and Thermo Scientific, respectively) were resuspended in Laemmli's 2x buffer, separated on SDS/PAGE and blotted onto nitrocellulose membrane (GE Healthcare, Little Chalfont, United Kingdom). Blots were incubated with the following antibodies (Abs): P-Y^694/699-STAT5, catalase (Cell Signaling Technology, Danvers, United States), STAT5 (BD Transduction Laboratories, Franklin Lakes, United States), actin (Santa Cruz, Dallas, United States), Flag M2 (Stratagene, Santa Clara, United States), Glutaredoxin1 (Glrx1) (R&D, Minneapolis, United States). Membranes were developed with the ECL chemiluminescence detection system (GE Healthcare, Little Chalfont, United Kingdom) using specific peroxidase (HRP) conjugated to rabbit or mouse IgG antibodies (Cell signaling Technology, Danvers, United States) or goat IgG (Santa Cruz, Dallas, United States).