Protein Extraction and Western Blot Analysis

Proteins were extracted using RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% Sodium Deoxycholate, 0.1% SDS and 50 mM Tris pH. 7,5) with 1 mM PMSF, 10 mM NaF, 2 mM NaVO₃ and Protease Inhibitor cocktail (cat.no. 04-693-1160-001, Roche, USA). For analysis of phosphorylated histones, extracts were prepared in boiling 2% SDS buffer (in 150 mM Tris pH 6,8). Western Blot (WB) analysis was performed as previously described in [17 (link)]. The following antibodies were used: mouse monoclonal antibodies (Mab) to p140Cap already characterized in [17 (link)] (1:500), anti phospho-p130Cas (Tyr410; #4011, 1:1000), anti phospho-Src (Tyr416; #2101, 1:1000), anti phospho-JAK2 (Tyr1007/1008; #3776S, 1:1000), anti γH2AX (Ser139; #2577, 1:1000) and anti H2AX (#2595, 1:1000) from Cell Signaling, Beverly, MA; anti GAPDH (MAB374, 1:8000) from Millipore, Billerica, MA, USA; anti p130Cas (cat.no 610272, 1:2500) from BD Transduction Laboratories, Franklin Lakes, NY; anti Src (B-12, 1:1000) and anti JAK2 (sc-278, 1:1000) from Santa Cruz Biotechnologies, Palo Alto, CA, USA; anti Tubulin (T5168, 1:8000) from Sigma-Aldrich Co, Italy. Secondary antibodies conjugated with peroxidase and nitrocellulose membranes were purchased from GE Healthcare (Buckinghamshire, UK). When appropriate, the membranes were stripped according to manufacturers’ recommendations and re-probed.