Downregulation of SHP protein by p53 is mediated by proteosome degradation.

<p>A: Western blots to determine the effect of p53 on SHPWT, SHPK170R, or SHPK170N protein expression. Flag-SHPWT, Flag-SHPK170R or Flag-SHPK170N expression vectors were transfected in 293T cells without or with Flag-p53 co-expression, and p53 and SHP proteins detected simultaneously using anti-Flag antibodies on the same blot. B: Immunoprecipitation and Western blots to determine the association of SHP proteins with p53 protein. Flag-SHPWT, Flag-SHPK170R or Flag-SHPG171A expression vectors were co-transfected with GST-p53 expression vector. Whole cell lysates were immunoprecipitated with the anti-GST antibodies, and SHP and p53 proteins were detected using anti-Flag or anti-GST antibodies, respectively. C: Western blots to determine the effect of p53 on SHPK170R protein levels. Flag-SHPK170R and Flag-p53 plasmids were cotransfected in 293T cells, and SHP and p53 proteins were detected using anti-Flag antibodies. p53 and SHP protein can be easily distinguished because of different molecular weight. D: Western blots to determine the effect of p53 on SHPG171A protein levels. Myc-p53 and Flag-SHPG171A plasmids were cotransfected in 293T cells, and p53 and SHPG171A proteins were detected using anti-Myc and anti-Flag antibodies, respectively. E–F: Western blots to determine the effect of p53 on SHP protein expression in the presence of MG132 or TSA (E) or on the phosphorylated and acetylated SHP protein expression (F). Flag-SHP expression vectors were transfected alone or together with Myc-p53 vectors, and both proteins were detected using antibodies as indicated in the figures. CON, control. G: <i>In vitro</i> Ubiquitination assays to determine the effect of p53 on SHP protein ubiquitination. Flag-SHP and HA-Ub expression vectors were transfected without or with GFP-p53 vectors. Whole cell lysates were immunoprecipitated with the anti-Flag antibodies, and ubiquitinated SHP was detected using anti-HA antibodies (indicated by a solid line).