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Figure S1. c-Myc and Fbw7γ half life is independent by p53. (a) WB analysis of c-Myc and β-actin protein levels (left) and c-Myc mRNA levels (right) in p53−/− and WT MEFs. Data represent the mean of three determinations ± SEM. (b) WB analysis of endogenous c-Myc protein stability in p53−/− and WT MEFs. Cells were treated with CHX and harvested at the indicated time points. β-tubulin was used as internal control. (c, left) WB analysis of c-Myc and NPM protein levels in WT and p53−/− MEFs infected with lentiviruses expressing siNPM-RNA or control (EV) lentiviruses. (right) c-Myc mRNA levels in the same cells evaluated by QPCR (results are normalized against EV values). (d) Immunofluorescence analysis of Fbw7γ and NPM localization in dKO MEFs expressing Flag-Fbw7γ (red staining) and infected with a GFP-NPM–expressing retrovirus (green staining). Nuclei were also stained with DAPI (merging of the Fbw7γ, NPM, and DAPI staining are shown). (e) WB analysis of HA-Fbw7γ protein stability in p53−/− and WT MEFs. Cells were infected with a HA-Fbw7γ–expressing retrovirus than treated with CHX and harvested at the indicated time points. As an internal control, β-tubulin was used. (f, top left) WB analysis of the Skp2 protein levels in dKO and p53−/− MEFs. (top right) Immunofluorescence analysis of Skp2 cellular localization in dKO and p53−/− MEFs (red staining). Anti-UBF antibodies have been used as nucleolar marker (green staining). Nuclei have been stained with DAPI (a merging of the Skp2, UBF, and DAPI staining is shown). (bottom) Whole cell lysate from p53−/− MEFs was immunoprecipitated with an anti-NPM antibody and an unrelated antibody as a negative control. Total lysates and immunoprecipitates were resolved by SDS-PAGE and blotted with antibodies against NPM or Skp2. (f) Western blot analysis of MEFs infected with a retrovirus expressing NPMmut or control (EV) retrovirus. Total cells lysates have been resolved on SDS-PAGE and analyzed with different antibodies as indicated. Numbers to the sides of the gel blots indicate molecular mass standards in kD. Bars, 10 µm.