Figure 3.

The Serine 18-Containing Isoform of USP7 Is Required for Mdm2 Stability and Concomitant p53 Downregulation

(A) N-terminal sequences of the isoforms of USP7, demonstrating the only isoform containing serine 18 (USP7S). Identical sequences are shaded.

(B) HeLa whole-cell extracts (Input, 30%) were immunoprecipitated using USP7S antibodies, and the pull-down (PD, 100%) or the unbound proteins (UP, 50%) were separated by western blotting with USP7 antibodies specific to Ser18-containing isoform and antibodies cross-reacting with all the isoforms of USP7 (USP7_total).

(C) HeLa cells were treated with Lipofectamine RNAiMAX transfection reagent in the absence (Control) and presence of USP7S specific siRNA (200 pmol) for a further 48 hr and then immunostained with pUSP7S antibodies followed by Alexa Fluor 488 anti-rabbit IgG.

(D) Cytoplasmic (C) and nuclear (N) fractions were prepared from HeLa cells and 40 μg of protein in the C fraction and an equal volume of the N fraction were separated by 10% SDS-PAGE and analyzed by western blotting with USP7S, pUSP7S, and USP7_total antibodies. Tubulin and fibrillarin were used as cytoplasmic and nuclear markers, correspondingly.

(E) HeLa cells were treated with Lipofectamine RNAiMAX transfection reagent in the absence (Control) and presence of USP7_total or Ser18-containing USP7-specific siRNA (200 pmol) for 72 hr. Cells were pelleted by centrifugation, and whole-cell extracts were prepared and analyzed western blotting.

(F) HCT116 p53^+/+ cells were treated with Lipofectamine RNAiMAX transfection reagent in the absence (Control) or presence of USP7S siRNA (200 pmol) for 72 hr. Cells were pelleted by centrifugation, cytoplasmic (C) and nuclear (N) fractions were prepared and 40 μg protein in the C and an equal volume in the N fraction were analyzed by western blotting.