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. 2011 Jan 31;6(1):e16598. doi: 10.1371/journal.pone.0016598

Figure 4. USP7 regulates PML protein levels and physically interacts with PML.

Figure 4

(A–C) CNE2 cells (A and B) or H1299 cells (C) were subjected to 2 rounds (+) or 3 rounds (++) of transfections with siRNA against USP7 (siUSP7) or GFP (siGFP) or no siRNA (mock). Equal amounts of total cell lysates were analyzed by western blotting using the antibodies indicated. In (C) the blot on the right confirms the lack of p53 expression in H1299 as compared to CNE2 cells. (D) CNE2 cells were treated with siRNA against USP7 or GFP then were transfected with a plasmid expressing HA-tagged ubiquitin. PML ubiquitylation was then analyzed by immunoprecipitating endogenous PML and western blotting with HA antibody. (E and F) Endogenous PML (E) or USP7 (F) was immunoprecipitated from CNE2 nuclear extracts using anti-PML or anti-USP7 antibody then western blotted using the reciprocal antibody as indicated. Normal rabbit IgG was used a negative control (IgG) for non-specific immunoprecipitation. ‘Input’ represents 5% of the nuclear lysate used for immunoprecipitation. The band representing IgG is marked by an asterisk.