Anti rabbit secondary antibody conjugated with horseradish peroxidase

The protein samples were separated by SDS-PAGE using 10 % acrylamide gel and transferred onto a polyvinylidene fluoride membrane. The membrane was incubated with 1 × TBS (Tris-buffered saline with 0.1 % (v/v) Tween 20) containing 6% (w/v) skim milk for 30 min. Then, the membrane was incubated with an anti-HA antibody (1:1000 dilution) at 4 °C overnight. After washing with 1 × TBS-T three times, the membrane was incubated with an anti-rabbit secondary antibody conjugated with horseradish peroxidase (1:5000 dilution) (Cell signaling Technology, cat no: 7074S) at 4 °C for 4 h. After washing with 1 × TBS-T three times, the membrane was immersed in ECL reagents (Thermo Fisher Scientific Inc., Waltham, MA, USA), and the chemiluminescence images were captured using the ChemiDoc^TM XRS+ imaging system (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

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).