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. 2008 Mar 21;105(12):4838–4843. doi: 10.1073/pnas.0712216105

Fig. 2.

Fig. 2.

Hh signaling promotes p53 ubiquitination and activates Mdm2. (a) RNA blotting, using total RNA from MEFs carrying the control vector and expressing Smo mutants, was carried out by using p53, Mdm2, Pirh2, and COP1 cDNA probes. 28s rRNA was examined as a loading control. (b) p53 and Smo expression plasmids were transfected into C3H10T1/2 cells, which were treated with 50 μM MG132 for 4 h before collection. Forty-eight hours after transfection, the amount of p53 was determined by immunoblotting. (c) C3H10T1/2 cells were transfected with expression plasmds for Flag-tagged ubiquitin, HA-p53, and the Smo mutant and treated with 50 μM MG132 for 4 h before harvesting. Twenty-four hours after transfection, ubiquitinated p53 was detected by immunoprecipitation with anti-HA antibody and immunoblotting with anti-Flag or anti-p53 antibody. Mono-ubiquitinated (Ub) and polyubiqutinated (Ub) p53 are indicated. (d) HA-tagged p53 expression plasmid was transfected into C3H10T1/2 cells, and the cells were incubated with 5 μg/ml Shh-N for 24 h. Ubiquitinated p53 was detected by immunoprecipitation with anti-HA antibody and immunobloting with anti-p53 (FL-393) antibody. (e) C3H10T1/2 cells were incubated with indicated amount of Shh-N for 48h. Ubiquitinated p53 was detected by immunoprecipitation with anti-p53 polyclonal antibody and immunobloting with anti-p53 monoclonal antibody. (f) The p53 expression vector was transfected into C3H10T1/2 cells with or without Smo mutants. p53 (red) was detected by using anti-p53 (FL-393) antibody; Smo (green) was visualized by using anti-gD antibody. The nucleus (blue) was stained with DAPI. (g) Quantification of p53 staining was performed as described in f. The staining pattern for p53 in cells expressing p53 and Smo mutants was scored for 100 cells in three separate experiments. The graph indicates the percentages of cells with the indicated p53 localization patterns. Bars indicate the SD of three independent experiments. (h) SMO-M1 or -M2 expression plasmid was transfected into C3H10T1/2 cells, and the cells were then treated with 50 μM MG132 for 4 h before harvest. Forty-eight hours after transfection, cell lysates were subjected to immunoprecipitation with anti-p53 antibody (FL-393) and immunoblotting with anti-p53 (Pab246) or anti-Mdm2 (2A10) antibody. (i) HA-tagged p53, mutant Smo, and Flag-tagged ubiquitin expression plasmids were transfected into GFP siRNA (Control)- and Mdm2 siRNA-expressing C3H10T1/2 cells. Twenty-four hours after transfection, detection of ubiquitination of p53 was performed as described in c. (j) Mutant Smo expression vectors were transfected into C3H10T1/2 cells. Cells were incubated with 15 μM Nutlin-3 (Calbiochem) for 16 h. p53 was detected by immunoprecipitation (IP), using anti-p53 (FL-393) antibody, followed by immunoblotting (IB) with a different anti-p53 (Pab246) antibody.