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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 8. — RFWD3 promotes interaction of ZRANB3 with polyubiquitinated PCNA and not ubiquitinated RPA. (A and B) HeLa cells (A) or U2OS cells (B) were transfected with siFF, siBRCA2-8, and/or siRFWD3-4 and then transfected 1 d later with His-ubiquitin. After one additional day, they were treated with 2 mM HU for 2 h. Cells were lysed under denaturing conditions, and His-tagged ubiquitinated proteins were purified by nickel beads and immunoblotted for endogenous RPA2. His-ubiquitin in lysates was detected by anti-His immunoblot. Asterisk represents phosphorylated RPA2 detected as a slower migrating band in RPA2 lysate immunoblots. (C) U2OS cells expressing HA-ZRANB3 were transfected with the indicated siRNAs and treated with UV radiation (30 J/m²). After 3 h, protein complexes were crosslinked with formaldehyde, and anti-HA immunoprecipitated proteins were immunoblotted for endogenous RPA2 and HA. (D) U2OS cells expressing HA-ZRANB3 were transfected with the indicated siRNAs and treated with ATRi (10 µM) for 30 min followed by UV radiation (30 J/m²) and incubation in ATRi (10 µM) for another 3 h. Protein complexes were crosslinked with formaldehyde, and anti-HA immunoprecipitated proteins were immunoblotted for endogenous PCNA and HA. Asterisk represents monoubiquitinated band of PCNA.