Western blot and protein lysate preparation

Protein was extracted from cell pellets using freshly prepared ice-cold urea lysis buffer (containing 6 M urea (Sigma, U0631), 25 mM Tris (pH 8.0) (Sigma, T2194), 1 mM EDTA (Sigma, E7889), 1 mM EGTA (Sigma, E0396), 1% phosphatase inhibitor cocktail 2 (Sigma, P5726), 1% phosphatase inhibitor cocktail 3 (Sigma, P0044), and 1% protease inhibitor cocktail (Sigma, P3840)). Lysis buffer was added directly to cell pellets (1 mL of lysis buffer per 5 × 10⁷ cells, or a minimum of 0.1 mL of lysis buffer for < 5 × 10⁶ cells), followed by two rounds of sonication (using a cup horn probe (Fisher Scientific, 550 Sonic Dismembrator) filled with ice water (30 s at 50% power), separated by a 10 s incubation on ice. The lysates were vortexed at maximum power for 15 s and centrifuged at 20,000 g for 10 min at 4°C to pellet the debris. The cleared lysate was then transferred to a fresh pre-cooled microcentrifuge tube and stored at −80°C. The protein concentration of the lysate was determined using the Pierce Micro BCA Protein Assay (Thermo, 23235).

Cell lysates were prepared for gel electrophoresis by diluting to a protein concentration of 1.2 ug/uL using 4X NuPAGE LDS Sample Buffer (Thermo, NP0007), 10X NuPAGE Sample Reducing Agent (Thermo, NP0009) and PBS (where the final concentration of the LDS Sample Buffer and Reducing Agent are 1X). Samples were heated at 98°C for 5 min, spun down (20,000 g for 10 s at room temperature), and 30 μg of total protein was loaded per well onto a polyacrylamide gel (NuPAGE 3%–8% Tris-Acetate Gels (Thermo, EA0375BOX) in 1X Tris-Acetate SDS Running Buffer (Thermo, LA0041) for CPT1A, CPT1B, CPT1C and CPT2 western blots; NuPAGE 4%–12% Bis-Tris Gels (Thermo, EA0321BOX) in 1X MES SDS Running Buffer (Thermo, NP0002) for GAPDH western blots). Proteins were then transferred to a membrane (Thermo, LC2001) using a traditional wet transfer with NuPAGE Transfer Buffer (Thermo, NP0006) and the XCell II Blot Module (Thermo, EI9051). Membranes were washed with 1X TBS (Cell Signaling, 12498S) for 5 min, blocked with 5% nonfat dried milk (Cell Signaling, 9999S) for 1 hour at room temperature, incubated with an antibody (dilution and solution according to manufacturer’s instructions) overnight at 4°C, washed three times with 1X TBST (Cell Signaling, 9997S), incubated with an anti-rabbit IgG HRP-linked secondary antibody (Cell Signaling, 7074) for 1 hour at room temperature, washed three times with 1X TBST and then incubated with a working solution of SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo, 34580) for 5 min at room temperature. Chemiluminescence on the membrane was detected using Bio-Rad’s ChemiDoc XRS+ Imaging System. Novex Sharp Pre-stained Protein Standard (Thermo Fisher Scientific, Cat # LC5800) was used in the molecular marker lane in western blots, with 80kDa and 40kDa MW marker band shown in related western. The following antibodies were obtained from commercial resources: recombinant anti-CPT1A antibody (Abcam, ab220789); recombinant anti-CPT1B antibody (Abcam, ab134135); recombinant anti-CPT2 antibody (Abcam, ab181114); CPT1C-specific antibody (Proteintech, Rosemont, IL, USA, 12969-1-AP); GAPDH antibody (Cell Signaling, 5174), phospho-Histone H2A.X (Ser139) antibody (Cell Signaling antibodies, 9718), Caspase-3 antibody (Cell Signaling antibodies, 9662), a-Actinin (D6F6) XP antibody (Cell Signaling antibodies, 6487), NRF2 antibody (Proteintech, 16396-1-AP), and histone H3 antibody (Cell Signaling Technologies, Inc. Boston, MA, USA, 17168-1-AP).