Ripa lysis buffer

The following agents were used in this study: sodium acetate, sodium propionate, and sodium butyrate were purchased from Sigma-Aldrich. SCFAs were a mixture of sodium acetate, sodium propionate, and sodium butyrate, which was dissolved in Tyrode’s buffer at a ratio of 3 mM sodium acetate, 1 mM sodium propionate, and 1 mM sodium butyrate. Both the rabbit anti-GPR41 and anti-GPR43 antibodies were purchased from Santa. The rabbit anti-GAPDH antibody was purchased from CST. The goat Anti-Rabbit IgG and the goat Anti-mouse IgG were purchased from Bio swamp. RIPA lysis buffer and the BCA protein assay kit were obtained from Bio swamp.

Treated cells were lysed with RIPA lysis buffer (Bioswamp), and the protein concentrations in the lysates were measured with a BCA protein assay kit (Bioswamp). Equal amounts of proteins in each sample were loaded onto gels and subjected to SDS-PAGE. The separated proteins on the gels were then transferred to nitrocellulose membranes. Each immunoblot was blocked with 5% nonfat milk in TBST for 2 h prior to an overnight incubation at 4 °C with specific antibodies. The following primary antibodies were used for Western blotting: Bcl-2 (Proteintech, 60178-1-Ig), Bax (Proteintech, 50599-2-Ig), cleaved caspase-3 (Cell Signaling Technology, 9664), LC3B (Bioswamp, MAB43969), Beclin 1 (Proteintech, 66665-1-Ig), Parkin (Proteintech, 14060-1-AP), P62 (Proteintech, 18420-1-AP), Tim23 (Proteintech, 11123-1-AP), CoxIV (Cell Signaling Technology, 4844) and NDP52 (Cell Signaling Technology, 60732). The blots were washed with TBST three times for 15 min each and then incubated with secondary antibodies for 2 h at RT. Finally, enhanced chemiluminescence reagent was used to visualize the bands, and the intensities were determined to further evaluate the levels of each protein.