Protein stability assay, western blotting, and antibodies

Cells were treated with siRNAs targeting USP1 and UAF1 for 48–72 hrs. 10 uM cycloheximide was then added for various durations of time (ex. 0, 3, 6 and 9 hrs). Cells were then prepared for western blot analysis and probed for RAD51AP1 protein levels. To test if RAD51AP1 is degraded by the proteasome, 10 uM MG132 was treated for the last 6 hrs of the 8 hr, 10 uM cycloheximide treatment. Cell extracts were run on an SDS-PAGE gel and then transferred to a PVDF membrane (Bio-Rad, Hercules, CA). Membranes were probed with primary antibodies overnight at 4C. The membranes were then washed and incubated with either mouse or rabbit secondary antibody linked to horseradish peroxidase (Cell Signaling Technologies). The bound antibodies were viewed with Pierce ECL Western Blotting Substrate (Thermo Scientific). The following antibodies were used: anti-RAD51AP1 (Abcam; cat#{"type":"entrez-nucleotide","attrs":{"text":"Ab101321","term_id":"32351915","term_text":"AB101321"}}Ab101321), anti-FANCD2, RAD51, PCNA antibodies (Santa Cruz Biotechnology), anti-RPA, phospho-RPA32 (S4/S8), USP1, WDR20, 53BP1 rabbit polyclonal antibodies (Bethyl Laboratory), anti-ubiquitin FK2 mouse antibody (Millipore), anti-γ-H2AX (Upstate), anti-γ-Tubulin, anti-FLAG antibodies (Sigma). Anti-UAF1 antibody generated from rabbit was previously described.^26,47