Chaps buffer

Cultured BMM were lysed in CHAPS buffer (Thermo Fisher Scientific) containing protein inhibitors. Ten to 40 μg of protein per sample was loaded to 10% SDS–polyacrylamide gel electrophoresis gels followed by transfer to polyvinylidene difluoride membranes. The membranes were then blocked with 5% nonfat milk in 1× tris-buffered saline plus 0.05% Tween 20 for 2 hours at room temperature and incubated with corresponding primary antibodies. Primary antibodies are as follows: anti-KDM5C (A301-034A, Bethyl Laboratories) or anti–β-actin (4967, Cell Signaling Technology) and OXPHOS rodent Western blots antibody cocktail (45-8099, Thermo Fisher Scientific). Blots were developed using the SuperSignalt West Dura Extended Duration Substrate (Thermo Fisher Scientific) and imaged using the ChemiDoc MP Imaging System (Bio-Rad).

Hippocampus extracts were prepared in 0.5% CHAPS buffer containing complete protease inhibitor (Thermo Scientific) and centrifuged for 15 min at 20,000g at 4 °C, and protein concentration was determined using a Pierce BCA Protein Assay Kit (Thermo Scientific). Supernatants were denatured in 5xLaemmli buffer, separated by SDS-PAGE gel electrophoresis, and transferred to nitrocellulose. Following blocking with Odyssey Blocking buffer (LI-COR Biosciences), the membrane was first incubated with primary Abs, and then with fluorophore-conjugated secondary Abs. Protein bands were visualized Odyssey Fc Imaging System (LI-COR Biosciences) and quantification was done in Image Studio (LI-COR Biosciences).

BMDMs (0.3 × 10⁶ cells/ml) were resuspended in 20 mM HEPES pH 7.5, 10 mM KCl, 1.5 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, 320 mM sucrose and EDTA-free protease inhibitor cocktail, and mechanically disrupted through 21 G needles 30 times. Lysates were centrifugated at 300×g 8 min and supernatants were lysed in 1 volume of CHAPS buffer (20 mM HEPES, 5 mM MgCl₂, 0.5 mM EGTA, 0.1% CHAPS and EDTA-free protease inhibitor cocktail) and then centrifugated at 5000 × g for 8 min. The pellets were washed once with PBS, resuspended in CHAPS buffer, crosslinked with disuccinimidyl suberate (DSS, 2 mM, 37 °C, 1 h) (Thermofisher), and then quenched in sample buffer 2X.