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. 2021 Jan 25;118(5):e2015339118. doi: 10.1073/pnas.2015339118

Fig. 3.

Fig. 3.

SIRT7 enhances association of NPM with HDM2 and prevents p53 ubiquitination under UV. (A) IF staining for NPM and HDM2 of SIRT7 KD and control U2OS cells 12 h after UVC irradiation. Nuclei were counterstained with DAPI (n = 3). (Scale bars, 25 µm.) (B) Coupled immunoprecipitation (IP) (HDM2 antibody) and Western blot analysis (NPM antibody) of SIRT7 KD and control (scrambled) U2OS cell lysates 3 h after UVC irradiation (n = 3). (C) Coupled IP (p53-DO-1 antibody) and Western blot analysis (ubiquitin antibody) of SIRT7 KD and control (scrambled) U2OS cell lysates 2 h after UVC irradiation followed by a 5-h incubation with 10 µM MG-132 (n = 3). The membrane was reprobed with anti-p53 antibody to ensure equal IP of p53 (Top). (D) Western blot analysis of p53 levels in WT and SIRT7 KO MEFs 12 h after UVC irradiation, followed by a 5-h treatment with 10 µM MG-132. RALA was used as loading control. Quantification of p53 relative levels ± SD is given (Bottom) (n = 3). (E) Western blot analysis of p53 levels in control (scrambled) and SIRT7 KD U2OS cell lysates 12 h after UVC irradiation followed by treatment with cycloheximide (CHX; 50 µg/mL). Quantification of p53 relative levels ± SEM is given (Bottom) (n = 3; two-way ANOVA). (F) Western blot analysis of p53, SIRT7, and NPM levels in NPM KD and control (scrambled) U2OS cells transfected with Flag-SIRT7 5 h after UVC irradiation. GAPDH was used as a loading control. Quantification of p53 relative levels ± SD is given (Bottom) (n = 3). *P < 0.05.