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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 9. — RFWD3 ubiquitin ligase activity promotes the polyubiquitination of PCNA. (A and B) U2OS cells were transfected with the indicated siRNAs, treated with 1 µg/ml 4NQO for 4 h, and endogenous PCNA ubiquitination was detected in whole cell lysates using an antibody against K164-ubiquitinated PCNA. (C and E) U2OS cells were transfected with the indicated siRNAs, treated with 30 µM ML323 for 4 h, and then treated with 1 µg/ml 4NQO in the presence of 30 µM ML323 for another 4 h. Endogenous PCNA ubiquitination was detected in whole cell lysates using an antibody against ubiquityl-K164 PCNA. (D) U2OS cells expressing siRNA-resistant RFWD3 (WT or C315A) were transfected with siRFWD3-4 and compared with U2OS cells transfected with siFF or siRFWD3-4. Cells were treated with 30 µM ML323 for 4 h and then with 1 µg/ml 4NQO in the presence of 30 µM ML323 for another 4 h. PCNA ubiquitination was detected using an antibody against K164-ubiquitinated PCNA. (F) FA patient fibroblasts complemented with WT RFWD3 (1143 + WT) or empty vector (1143 + mock) were treated with 1 µg/ml 4NQO for 4 h. PCNA ubiquitination was detected in whole cell lysates using an antibody against ubiquityl-K164 PCNA.