Western Blot Analysis of SHARPIN Protein

Western blot analysis was performed as previously described (16 (link)). Briefly, cell lysates were separated by SDS-PAGE and transferred to a PVDF membrane (Millipore, Billerica, MA, USA). The blot was subsequently probed with anti-SHARPIN (abs134288, ABSIN, Beijing, China) at a dilution of 1:500 or anti-GAPDH (Multisciences Biotech Co., Hangzhou, China) at a dilution of 1:5000, followed by incubation with a horseradish peroxidase-conjugated secondary antibody. The signal was detected using enhanced chemiluminescence (Millipore, Billerica, MA, USA). The expression level was quantified using the ImageJ program (NIH).