Western Blotting of Cell Signaling Proteins

Whole-cell extracts were prepared using 8 M UREA 0.5% Triton-X lysis buffer and 10–75 μg total protein was loaded onto denaturing polyacrylamide gels (Bio-Rad). Blots were incubated with the primary antibodies in TBS 0.05% Tween-20/ 5% milk or 5% BSA overnight at 4 °C, incubated with HRP coupled secondary goat anti-rabbit and goat anti-mouse antibody (Cell signaling) at 1:5,000 for 1 h at room temperature. Primary antibodies used were anti-HK1 (Cell Signaling, #2024, 1:1000), anti-HK2 (Cell signaling, #2867, 1:1000), anti-HK3 (Thermo Fisher Scientific, #PA5-29304, 1:500), anti-yH2AX Ser139 (Cell Signaling, #2577, 1:1000), anti-cleaved Caspase-3 (Cell Signaling, #966, 1:1000 Milk/TBS-T), anti-Puma (Cell Signaling, #12450, 1:1000 BSA/TBS-T), anti-Noxa (Enzo Life Sciences, ALX-804-408-C100, 1:1000 Milk/TBS-T), anti-BCL-2 (Santa Cruz, sc-509, 1:1000 BSA/TBS-T) and anti-BIM (Cell Signaling, #2933, 1:1000 BSA/TBS-T). Cytoplasmic and nuclear fractionation was performed as described before [25 (link)]. Uncropped western blots are shown in the supplement.