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. 2010 Nov 29;191(5):953–966. doi: 10.1083/jcb.201006043

Figure 5.

Figure 5.

Rad18 S box phosphorylation is UV-inducible and promotes interactions with Polη. (A) H1299 cells expressing HA-Rad18 were treated with UV (100 J/m2). After 1 h, extracts from the resulting cells were immunoprecipitated with anti-HA and analyzed by SDS-PAGE and immunoblotting with anti-HA and anti-Cdc7 antibodies. (B) HA-Rad18–expressing H1299 cells were transfected with siChk1 (to ablate Chk1 expression) or with siCon for controls. 24 h after transfection, cells were treated with UV (10 J/m2), and 1 h later, extracts were immunoprecipitated and analyzed as described for A. (C) H1299 cells were infected with adenovirus vectors encoding HA-tagged Rad18 (WT) or AdCon. Cells were treated optionally with UV (100 J/m2). After 1 h, extracts from the resulting cells were immunoprecipitated with anti-HA, and the immune complexes were analyzed by SDS-PAGE and immunoblotting. Immunoblots were probed sequentially with anti-Rad18 (pS434) and anti-HA. Input fractions were probed with a Chk1 (p317) antibody. (D) H1299 cells were coinfected with adenoviruses encoding HA-Rad18 (WT) and YFP-Polη, then transfected with siRNA against Cdc7 (or with nontargeting siCon RNA duplexes). Some cultures were treated with 100 J/m2 UV as indicated. One h after UV treatment, cells were harvested and the resulting extracts were immunoprecipitated with anti-HA. Immune complexes and input fractions were analyzed by SDS-PAGE and immunoblotting with anti-GFP (to detect YFP-Polη), anti-Rad18, and anti-Cdc7, as indicated. (E) H1299 cells were infected with adenoviruses encoding HA-Rad18 (WT), HA-Rad18 S-Box-A, or HA-Rad18 S-Box-D, or with AdCon. 48 h after infection, cells were harvested and the resulting extracts were immunoprecipitated with anti-HA. Immune complexes and input fractions were analyzed by SDS-PAGE and immunoblotting with anti-Polη, anti-Rad18, and anti-Rad6 as indicated. (F) H1299 cells were infected with adenoviruses encoding HA-Rad18 (WT) or HA-Rad18 S-Box-A, or with AdCon. Some cultures were treated with 100 J/m2 UV as indicated. 1 h after UV treatment, cells were harvested and the resulting extracts were immunoprecipitated with anti-HA. Immune complexes were analyzed by SDS-PAGE and immunoblotting with anti-Polη, anti-Rad18, and anti-Rad6 as indicated. (G) HCT116 RAD18−/− cells were infected with adenoviruses encoding HA-Rad18 (WT) or HA-Rad18 S-Box-A, or with AdCon. The resulting cells were treated with UV (0, 10, and 100 J/m2) as indicated. 1 h after UV treatment, cells were harvested and the resulting cell extracts were analyzed by SDS-PAGE and immunoblotting with anti-HA and anti-PCNA as indicated. Molecular mass is indicated in kilodaltons next to the gel blots.