Phospho histone γh2ax

H460 cell lines were treated with vehicle (DMSO) or ganetespib (25 nM, 50 nM) with or without irradiation (2 Gy, 4 Gy) as described above and were harvested 24 hours after genetespib treatment. After washing with ice-cold PBS, cell lysates were collected in RIPA Buffer (Life Technologies, Grand Island, NY, USA) with protease inhibitor cocktail (Roche, Eugene, OR USA). Protein concentrations were measured by protein assay kit (Life Technologies, Grand Island, NY, USA). Total cellular lysates were loaded onto 4–12% Bis-Tris gel (Bio-Rad, Hercules, CA, USA), separated by electrophoresis and electro-transferred onto nitrocellulose membranes. Membranes were blocked for 1 h at room temperature in 5% nonfat dry milk in Tris-Buffer saline containing 0.1% Tween20 (TBST). The membrane was incubated with primary antibodies (p21, phospho-CDC2, CDC2, phospho-p90RSK, p90RSK, phospho-AKT, AKT, phospho-mTOR, mTOR, phospho-EGFR, EGFR, pS6, cleaved caspase 9 and caspase 7, phospho-histone γH2AX, GAPDH, β-actin antibodies are all from Cell Signaling Technology, Danvers, MA, USA) at 4 °C overnight and subsequently incubated with a secondary antibody for 1 hour at room temperature. After washing the membrane with TBST, HRP activity was detected using Clarity Western ECL substrate (Bio-Rad, Hercules, CA, USA) and analyzed by ChemiDoc imager (Bio-Rad, Hercules, CA, USA).