Anti hexokinase 1

Additional validation experiments were performed using immunoblots. Protein lysates (50 g per lane), as determined by bicinchoninic acid assay (BCA) (Pierce) from two biological replicate animals each from compartments S1, S2, and S3 for vehicle and 50 mg/kg paraquat hearts were denatured by boiling and resolved on 12% sodium dodecyl sulfate-polyacrylamide gels. Proteins were then transferred to polyvinylidene fluoride membranes and stained with Ponceau S (Sigma). After de-staining, membranes were blocked in 5% bovine serum albumin at $22{}^{\circ }\text{C}$ for 2 h and then probed with either anti-Hexokinase II (Cell Signaling Technology, cat. #2867, 1:1000 dilution) or the negative control anti-Hexokinase I (Cell Signaling #2024, 1:1000 dilution) at $22{}^{\circ }\text{C}$ for 2 h, followed by anti-rabbit IgG, HRP-linked secondary antibody (Cell Signaling #7074, 1:1000 dilution) at $22{}^{\circ }\text{C}$ for 2 h. Pierce ECL Plus western blotting substrate (Thermo) was used to detect targeted proteins using Typhoon 9400 Imager (Molecular Dynamics). The ImageJ software (National Institutes of Health, Bethesda, MD, USA)^{63 (link)} was used to quantify the intensity of the bands of interest and normalized to Ponceau Stain^{64 (link)}. Statistical tests on the densitometry values were performed using Welch’s t-test in R v.3.6.3.

Cells were harvested and lysed with RIPA buffer containing a protease inhibitor cocktail (Roche) for IB analysis. The following antibodies were used for IB analysis: anti-ARF-BP1 antibody (1:1000–Abcam), anti-Hexokinase 2 (1:2000–Santa Cruz), anti-Hexokinase 2 (1:1000 –Cell Signaling), anti-Hexokinase I (1:1000–Cell Signaling), anti-α-Tubulin (1:10,000–Sigma), anti-GRP78 (1:10,000–BD Sciences), anti-β-Actin (1:10,000–Sigma), anti-Flag (1:1000–Sigma), anti-GFP (1:1000–Cell Signaling), anti-Myc (1:2000–Roche), anti-HA (1:1000– Sigma), anti-p53 (1:10,000–Santa Cruz), anti-K63-Ub (1:1500–Cell Signaling), anti-K48-Ub (1:1,000–Cell Signaling) and anti-His (1:1000 –Santa Cruz). For IP, cells were lysed with E1A lysis buffer (250 mM NaCl, 50 mM HEPES, pH 7.5, 0.1% NP40, 5 mM EDTA) and a protease inhibitor cocktail (Roche). Cell lysates were immunoprecipitated with anti-HA antibody (Sigma), anti-HK2 antibody (Santa Cruz) and anti-Lasu1 (HectH9; Bethyl Lab), according to manufactory’s instructions. Detailed antibody information is listed in Supplemental Table 1. Uncropped scans for blots are presented in Supplementary Fig. 12.