Figure 2.

Damage-inducible Rad18–SHPRH binding coincides with loss of high molecular weight Rad18. (A) Inhibition of checkpoint kinases does not affect the Rad18–SHPRH interaction. Cells transfected with FLAG-Rad18 and GFP-SHPRH were mock treated or exposed to 0.005% MMS and the respective kinase inhibitors for 4 h before being lysed and analyzed as in Fig. 1 B. ATRi, ATR inhibition; ATMi, ATM inhibition. (B) Rad18 and SHPRH interact specifically after MMS or H₂O₂ treatment. Cells expressing FLAG-Rad18 and GFP-SHPRH were treated with 50 ppm MMS, 50 J/m₂ UV, 30 µM mitomycin C (MMC), 0.1% ethyl methanesulfonate (EMS), 20 µM 4-NQO, 30 µM aphidicolin (Aph), 2 µM camptothecin (CPT), 1 µM actinomycin D (ActD), 20 µM etoposide (Etop), 0.1% hydrogen peroxide (H₂O₂), or 50 µM cis-platinum for 4 h before being lysed under condition B and analyzed by Western blotting. (C) Endogenous Rad18 is deubiquitinated after MMS and H₂O₂ treatment. Untransfected cells were treated with UV (0, 100, 200, and 400 J/m²), MMS (0, 25, 50, and 100 ppm), or H₂O₂ (0, 1, 2, and 4 mM) for 4 h before being lysed and analyzed by Western blotting.