Secondary hrp labeled antibodies

Total cell lysates were prepared with the Mammalian Cell Extraction kit (MBL, Watertown, MA), supplemented with 1 mM sodium orthovanadate. Protein quantification was performed with the DC Protein Assay (Bio-Rad). Forty micrograms of total cellular proteins was resolved on 12.5% sodium dodecyl sulfate–polyacrylamide gels and transferred on polyvinylidene difluoride membranes (GE Healthcare, Milan, Italy). The following antibodies were used: phospho–AKT-1 (Ser473), AKT, phospho-GSK3β (pGSK3β; Ser9), GSK3β, phospho–5' adenosine monophosphate (AMP)-activated protein kinase (pAMPK; Thr172), AMPK, full-length 116-kDa and 89-kDa cleavage fragment of PARP (Asp214), phospho-ERK1/2, ERK1/2, Nrf2 (all from Cell Signaling Technology, Beverly, MA); HO-1, GSH peroxidase, Cu/Zn superoxide dismutase (SOD), G6PD (Stressgen, Enzo Life Sciences, Milano, Italy); glutamate-cysteine ligase, catalytic subunit (GCLC) (Abnova, c/o EMBL Enterprise Management Technology Transfer GmbH (EMBLEM), Heidelberg, Germany); HRP-conjugated anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Novus Biologicals Inc., Littleton, CO); secondary HRP-labeled antibodies (Cell Signaling Technology). The ECL-Plus reagent was from GE Healthcare.

Western blots were performed using standard methods using a Reinhard lysis buffer supplemented with protease and phosphatase inhibitors (Roche, Basel, Switzerland). For phosphorylation studies, BaF3 and 32D cells were depleted of IL-3 for 15 h. Proteins were blotted on a nitrocellulose membrane. Primary antibodies were used at 1:1000 in 1% milk over night at 4 °C against phosphorylated and non-phosphorylated STAT1 (pY701), STAT3 (pY705 and pS727), STAT5 (pY694), STAT6 (pY641), JAK2, PLCγ1 (pY783), PLCγ2 (pY759), SLP76, ERK (T202/Y204), AKT (S473), SYK, ITK (all Cell Signaling, Danvers, MA, USA), JAK2, TEL (Santa Cruz Biotech, Dallas, TX, USA), SLP76 (pY128) (BD Pharmingen), and β-Actin (Sigma-Aldrich). Secondary HRP-labeled antibodies (Cell Signaling Technology) were used at 1:2500. Membranes were incubated with ECL (Amersham, London, UK) for 2 min and chemiluminescence was detected using a western blot detection system (Curix 60, AGFA).