FIGURE 3.
C1orf124 participates in the PRR pathway. A, C1orf124 is required for UV light-induced PCNA ubiquitination. HeLa cells with stable knockdown of C1orf124 were transfected with a control vector or reconstituted with shRNA-resistant (shR) constructs encoding full-length (FL) C1orf124, C1orf124ΔPIP, or C1orf124ΔUBZ. Cells were treated with 100 J/m2 UV light and collected 4 h later. The levels of unmodified PCNA and Ub-PCNA were analyzed by immunoblotting with anti-PCNA antibody. WB, Western blot. B, C1orf124 interacts with RAD18. 293T cells were transfected with plasmids encoding SFB-tagged RAD6, RAD18, and PCNA together with plasmids encoding Myc-tagged C1orf124. Coprecipitation was carried out using S-protein beads, and immunoblotting was performed using the indicated antibodies. C, C1orf124 regulates the chromatin association of RAD18. HeLa cells with stable expression of control (shControl) and C1orf124 (shC1orf124) shRNAs were left untreated or treated with 10 J/m2 UV-C light and incubated for 6 h prior to immunostaining with the indicated antibodies. CPD, cyclobutane pyrimidine dimer. D, C1orf124 regulates the chromatin association of RAD18. HeLa cells with stable expression of control and C1orf124 shRNAs were left untreated or treated with 100 J/m2 UV-C light and incubated for 4 h. Soluble and chromatin-bound RAD18 levels were analyzed by immunoblotting with anti-RAD18 antibody. E, C1orf124 and RAD18 function in the same PRR pathway. C1orf124-depleted cells, RAD18−/− cells, and cells depleted of both RAD18 and C1orf124 were treated with increasing doses of UV light. Survival curves are shown for the indicated cell lines. Data are presented as means ± S.D. from three different experiments.