Western blotting was conducted as previously reported(Rashad et al., 2022 (link); Rashad et al., 2020a (link)). Briefly, for the analysis of puromycin incorporation assay, cells were incubated in a complete growth medium with 10 μg/ml puromycin then homogenized in T-PER™ tissue protein extraction reagent (Thermo Fisher Scientific, Cat# 78510) with Triton(R) X-100 (Nacalai Tesque, Cat# 35501-15) and cOmplete™ protease inhibitor cocktail (Roche, Cat# 4693116001). Proteins were isolated from the supernatant, quantified using the Brachidonic-acid assay kit (BCA) (Thermo Fisher Scientific, Cat# 23227), separated on Mini-PROTEAN TGX gel (Bio-Rad, Cat# 4561096), and transferred to Polyvinylidene difluoride membrane (PVDF) (Bio-Rad, Cat# 1704156). Membranes were blocked in 5% Skim milk powder (Fujifilm, Cat# 190-12865) with Phosphate buffered saline with tween® (PBS-T) (Takara, Cat# T9183). The primary antibody was incubated overnight at 4°C, followed by room temperature incubation with the secondary antibody (IgG detector solution v2). Signal detection was performed using Pierce ECL substrate reagent (Thermo Fisher Scientific, Cat# 32106) on a ChemiDoc machine (Bio-Rad). To normalize protein expression, membranes were stripped with Restore™ stripping buffer (Thermo Fisher Scientific, Cat# 21059) and reprobed with Anti-beta actin antibody.
List of antibodies used: