α rpa194

Following 72 h of siRNA depletion, cells were collected, and total protein was harvested and prepared as in (Farley-Barnes et al. 2018 (link)). A Bradford assay (Bio-Rad 5000006) was used to quantify amount of total protein. An amount of 25–50 µg of total protein was separated by SDS-PAGE and transferred to a PVDF membrane (Bio-Rad 1620177). Membranes were blocked for 1 h in 5% milk in 1× PBST (or 5% BSA in 1× PBST for western blots of immunoprecipitations) and incubated overnight with the specified primary antibodies at 4°C. Proteins were detected with the following antibodies: α-RSL24D1 (dilution 1:1000; Proteintech 25190-1-AP), α-RPA194 (dilution 1:1000; Santa Cruz sc-48385), α-WDR12 (1:1000; Bethyl Laboratories A302-650A), α-PES1 (1:1000; Bethyl Laboratories A300-902A), α-p53 (dilution 1:5000; Santa Cruz sc-126), α-p21 (dilution 1:400, Santa Cruz sc-6246), α-puromycin (dilution 1:10,000; Kerafast EQ0001), and α-β-actin (dilution 1:30,000; Sigma Aldrich A1978). The western blots were developed with enhanced chemiluminescence reagents (Thermo Scientific 34096). Images were acquired by digital imaging using the Bio-Rad ChemiDoc Imaging System. Images were quantified with ImageJ software.