Western Blot Analysis of Protein Samples

Western blotting was conducted as described previously with some modifications [27 (link)]. Briefly, cells were lysed with an appropriate amount of RIPA lysis buffer (Beyotime, P0013B) on ice and centrifuged at 13,000 rpm for 15 min at 4 °C. The supernatants were collected and processed with 5 × SDS-PAGE loading buffer. The protein concentration of all samples was measured using the BCA assay (Thermo Scientific, 23,227). Protein samples (about 30 μg) were separated on 10% SDS-gel electrophoresis and transferred to PVDF membranes (Merck Millipore, ISEQ00010). Then, the membranes were incubated with the primary antibody overnight at 4 °C, followed by Alexa Fluor-conjugated secondary antibodies. Detection was performed using the Odyssey Infrared Imaging System (LI-COR, Biosciences, Lincoln, NE, USA).