“…Sorted cells grown in the presence of growth factor and those selected for factor independence were then starved from serum and cytokines for 5 hours and lysed in 1% NP40 lysis buffer (containing 100 mM Na orthovanadate, 100 mM phenylmethylsulfonyl fluoride, and a cocktail of protease inhibitors), as described 42 and processed for Western blotting with antibodies against JAK2, STAT3, Erk1/2, or against phosphosites in these proteins that reflect activation. 43 The sources of antibodies were: anti-HA (Roche Diagnostics, Indianapolis, IN), anti-JAK2 (Santa Cruz, anti-actin, Sigma, St. Louis, MO), anti-phospho-JAK2 (Tyr1007/1008), anti-phospho-STAT1 (Tyr701), anti-phospho-STAT3 (Tyr705), anti-phospho-STAT5 (Tyr694), anti-phospho-extracellular signalregulated kinase 1/2 (Erk1/2; Tyr202/204), and antibodies recognizing Erk1/2 were obtained from Cell Signaling Technology Inc. (Danvers, MA).…”