SHP destabilizes p53 protein by enhancing the activity of Mdm2.

<p>A: Western blots. Plasmids expressing Flag-p53 were co-transfected with Flag-SHP plasmids in 293T or Hela cells. p53 or SHP protein levels were determined using anti-Flag antibodies. B: Western blots. Flag-SHP and Myc-p53 plasmids were cotransfected in Hela cells, and SHP and p53 proteins were detected using anti-Flag and anti-Myc antibodies, respectively. C: Western blots. Myc-p53, Flag-SHP or HA-Mdm2 expression vectors were transfected in Hela cells, and Mdm2, SHP and p53 proteins were detected using anti-HA, anti-Flag or anti-Myc antibodies, respectively. D: Ubiquitination assays. Hela cells were cotransfected with GFP-p53, HA-Mdm2, and various Flag-SHP expression vectors together with Myc-ubiquitin (Myc-Ub) plasmids. GFP-p53 was immunoprecipitated from cell extracts with anti-GFP antibodies, and ubiquitinated p53 in the immunoprecipitates was detected by Western blots with anti-Myc antibodies. Positions of ubiquitinated p53 proteins are indicated by a solid line. E: Western blots. HA-Mdm2, Flag-p53 or Flag-SHP expression vectors were transfected in 293T cells, and Mdm2, SHP and p53 proteins were detected using anti-HA or anti-Flag antibodies, respectively. F: Ubiquitination assays. 293T cells were cotransfected with Flag-p53 and GFP-SHP expression vectors together with HA-ubiquitin (HA-Ub) plasmids. Flag-p53 was immunoprecipitated from cell extracts with anti-Flag antibodies, and ubiquitinated p53 in the immunoprecipitates was detected by Western blots with anti-HA antibodies. Positions of ubiquitinated p53 proteins are indicated by a solid line. G: Ubiquitination assays. 293T cells were cotransfected with GFP-p53, HA-Mdm2, and various Flag-SHP expression vectors together with Myc-ubiquitin (Myc-Ub) plasmids. GFP-p53 was immunoprecipitated from cell extracts with anti-GFP antibodies, and ubiquitinated p53 in the immunoprecipitates was detected by Western blots with anti-Myc antibodies. Positions of ubiquitinated p53 proteins are indicated by a solid line.