Anti hk1

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.

For western blot analysis, 2 × 10⁶ cells were plated on 10 cm plates and allowed to grow for 24 hr. The cells were then treated as described in the figure legends or harvested in PBS, and cell pellets were washed and frozen at −80°C. Cell extracts were then made using ice-cold lysis buffer [20 mM Hepes, 1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 10 mM sodium pyrophosphate, 100 mM NaF, 5 mM iodo-acetic acid, 20 nM okadaic acid, 0.2 mM phenylmethylsulfonyl fluoride and a complete protease inhibitor cocktail tablet (Thermo Fisher)]. For the tissue extracts, frozen tissues collected by liquid nitrogen snap freezing were thawed and homogenized in the same buffer. The extracts were run on 6% to 12% SDS-PAGE gels, transferred to PVDF membranes, and probed with the following antibodies: anti-phospho-Akt Ser473, anti-panAkt, anti-cleaved caspase-3, anti-HK1, anti-HK2 anti-PTEN (Cell Signaling Technology, Danvers, MA), anti-HA (Covance, San Diego, CA), anti-4HNE (JaICA, Japan), anti-catalase, anti-CuZnSOD and anti-MnSOD (StressGen, Farmingdale, NY), anti-SESN3 (ProteinTech, Rosemont, IL), and anti-ß-actin (Sigma). Immunoblots were quantified using the NIH ImageJ software program by densitometric signal and normalized as described in figure legends.

Protein samples were separated on NuPAGE Bis-Tris polyacrylamide gels (ThermoFisher, Waltham, MA, USA) and transferred to PVDF membranes (GE Healthcare, Chicago, IL, USA). The membranes were blocked in 5% BSA in PBS with 0.1% Tween-20 and incubated overnight at 4 °C with the following primary antibodies: anti SOD1 (Cell Signaling, Danvers, MA, USA, 1:1000), anti VDAC1 (Abcam, Cambridge, UK, 1:1000), anti HK1 (Cell Signaling, 1:1000), anti β-Tubulin (Cell Signaling, 1:2000), anti COX IV (Cell Signaling, 1:1000), anti SDHA (Abcam, 1:1000), anti β-Actin (Cell Signaling, 1:5000), anti Caspase-3 (Cell Signaling, 1:1000), and anti-cleaved Caspase-3 (Cell Signaling 1:1000). Membranes were incubated with IRDye conjugated secondary antibodies (LI-COR Biosciences, Lincoln, NE, USA, 1:25.000). Signals were detected using Odissey Imaging System (LI-COR Biosciences). Band quantification was performed by densitometric analysis using Image Studio Lite software (version 5.2.5, LI-COR Biosciences).