Figure 2.

Phosphorylation of Serine 18 Regulates USP7S Stability and Activity

(A) HeLa cells were treated with Lipofectamine RNAiMAX transfection reagent in the absence (Control) and presence of CK2α- or CK2α′-specific siRNA (200 pmol) for 72 hr. Cells were pelleted by centrifugation, and whole-cell extracts were prepared and analyzed by western blotting with indicated antibodies.

(B) HeLa cells were treated with Lipofectamine transfection reagent in the absence (Control) and the presence of USP7S or USP7S18A expressing plasmid (0.3 pmol) for 24 hr. Cells were pelleted by centrifugation, and whole-cell extracts were prepared and analyzed and western blotting.

(C) HeLa cells were treated with Lipofectamine transfection reagent in the presence of USP7S (0.2 pmol) or USP7S18A (0.6 pmol) expressing plasmid for 24 hr. Cells were then treated with 50 μg/ml cycloheximide for 0, 1, 2, or 4 hr and pelleted by centrifugation. Whole-cell extracts were prepared, and the amounts of USP7 were analyzed by western blotting. Statistical data are presented as a mean ± SD of three independent biological experiments.

(D) HeLa cells were treated in the presence of CK2α- or CK2α′-specific siRNA as described above, total RNA was prepared and the levels of USP7S and actin mRNA were analyzed by quantitative rtPCR. Statistical data are presented as a mean ± SD of three independent biological experiments.

(E) HeLa cells were cotransfected with 1.0 pmol of ubiquitin and 0.5 pmol of either USP7S or USPS18A expressing vectors. After 24 hr, cells were treated with 10 μM MG-132 for 6 hr and pelleted by centrifugation. Equal amounts of whole-cell extracts (500 μg) were then used to pull down Flag-tagged USP7S or USP7S18A using Flag-agarose beads. Immunoprecipitates were using ubiquitin or Flag antibodies.

(F) HeLa cells were treated with Lipofectamine transfection reagent in the absence (lane 1) or presence of 0.7 pmol of Mdm2 and 0.2 pmol of either USP7S or USPS18A expression vectors (lanes 3 and 4, respectively). After 24 hr, cells were pelleted by centrifugation and whole-cell extracts were prepared and analyzed by western blotting.

(G) Alternatively, HeLa cells were transfected with a 3-fold excess of USP7S18A (0.6 pmol) relative to USP7S expression plasmid.

(H) U2OS cells were treated with Lipofectamine transfection reagent in the absence (lane 1) or presence of 0.45 pmol p53, 0.55 pmol Mdm2, and 0.1 pmol USP7S, USPS18A, or USP7ΔN expression plasmids. After 24 hr cells were pelleted by centrifugation, and whole-cell extracts were prepared and analyzed by Western blotting.