Figure 1.

USP7 regulates H2A and γH2AX ubiquitination in DNA damage response. (A) HCT116 and HCT116-USP7^−/− cells were exposed to 20-J/m² UV and the cell extracts were prepared at indicated time points following UVR. The uH2A and ubiquitinated γH2AX were examined by Western blotting using anti-uH2A and γH2AX antibodies. (B) The cells were treated as in Figure 1A. Ring1B and Bmi1 were examined by Western blotting. (C) The cells were subjected to UVR and then treated with or without proteasome inhibitor MG132 and the cell extracts were examined by Western blotting. “Long Exp." indicates longer exposure for chemiluminescent detection (D) Anti-γH2AX blots were overexposed to show low level of γH2AX ubiquitination in HCT116-USP7^−/− cells. (E) HeLa cells were transfected with USP7 siRNA or control siRNA for 2 rounds. The transfected cells were irradiated with UV and processed as in Figure 1B. Ubiquitinated γH2AX, uH2A, USP7 and other DDR factors were examined by Western blotting. (F) HCT116 cells were exposed to UVR, and then incubated with USP7 inhibitor HBX 411108 at indicated concentration for 8 h. The Ub-γH2AX/γH2AX ratio is calculated based on gray scale of the blots examined by ImageJ.