Nb600 532ss

Total protein was extracted from cells using RIPA buffer (Beyotime, Shanghai, China) supplemented with a protease inhibitor cocktail (BioDee, Beijing, China) (100:1) and phenylmethylsulfonyl fluoride (Beyotime, Shanghai, China) (100:1). Proteins were separated on 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels (GenScript, Nanjing, China). The proteins were transferred to polyvinylidene difluoride membranes, and then blocked with 5% non-fat milk for 1 h. The membranes were incubated with the primary antibodies CPT2 (1:1,000, NBP1-32226; Novus, Columbia, USA) and beta-actin (1:5,000, NB600-532SS; Novus, USA) for 1 h, and washed with PBST (Solarbio, China) three times, for 5 min each time. The membranes were then incubated with anti-rabbit secondary antibody conjugated with horseradish peroxidase (1:1,500, 7074; Cell Signaling Technology, Boston, MA, USA) for 1 h at room temperature, and washed three times using PBST. Protein bands were visualized using an enhanced chemiluminescence system (GE Healthcare, Fairfield, CT, USA).

Cells were lysed in ice-cold RIPA buffer supplemented with Pierce™ protease and phosphatase inhibitor mini tablets (Thermo Scientific). Protein concentration was measured using the Pierce™ Rapid Gold BCA Protein Assay Kit and 30–40 µg protein samples were run on 10% SDS gels for protein separation, followed by blotting the gels on 0.2 µm nitrocellulose blotting membrane (Amersham, Freiberg, Germany) at 300 mA for 1 h in a cold room. After blotting, membranes were blocked with 5% skimmed milk for 1 h. Antibody concentrations used were as follows: β-actin, 1:10,000 (NB600-532SS, Novus Biologicals); LC3, 0.2 µg/mL (L8918, Merck) [31 (link)].