Quantifying USP48 and GAPDH Levels by Western Blotting

USP48 and GAPDH protein levels were analyzed by western blotting [24 (link)]. Briefly, KNG cells were lysed, and 30 µg of protein was separated by 10% SDS-PAGE and transferred onto polyvinylidene fluoride membranes. Subsequently, all membranes were blocked with 5% skimmed milk diluted in TBS at 37˚C for 1 h and washed thrice with TBST. The membranes were then incubated with rabbit polyclonal USP48 (dilution 1:500, ab237765, Abcam, San Diego, CA, USA) and rabbit monoclonal antibodies against GAPDH (dilution 1:10,000, AF7021, Affinity) at 4˚C for 12 h. The membranes were washed thrice with TBST. Secondary antibodies were detected using Goat Anti-Rabbit IgG H&L (HRP) (dilution, 1:10,000, ab205718, Abcam) and visualized using an enhanced chemiluminescent reagent (PerkinElmer Life Sciences, MA, USA).