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. 2023 Apr 10;222(5):e202106022. doi: 10.1083/jcb.202106022

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© 2023 Moore et al.

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Figure S4. — Analysis of RPA2 ubiquitination and phosphorylation in BRCA1/2-deficient cells. (A and B) U2OS cells were transfected with the indicated siRNAs and then transfected 1 d later with His-ubiquitin. After one additional day, they were treated with 2 mM HU for 2 h and lysed under denaturing conditions. His-tagged ubiquitinated proteins were purified by nickel beads and immunoblotted for endogenous RPA2. His-ubiquitin in lysates was detected by anti-His immunoblot. Asterisks represent phosphorylated RPA2 detected as a slower migrating band in the RPA2 lysate immunoblots. (C) U2OS cells were transfected with siFF, siBRCA2-3, and/or siMUS81 and treated with 4 mM HU for 4 h. Phosphorylated RPA2 was detected in lysates using the indicated phospho-specific antibodies. Asterisk is the same as in Fig. S4, A and B. (D and E) Sensitivity of U2OS cells to camptothecin (CPT) upon RFWD3 depletion using the indicated siRNAs. Cell survival is normalized to the untreated control for each siRNA. Data represent the mean and SD of three replicates per CPT dose and siRNA condition. Asterisks indicate P values for siRFWD3 versus siFF using an unpaired t test (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). Immunoblot shows RFWD3 depletion for each siRNA.