TSC22 inhibits HDM2- and E6-mediated p53 ubiquitination.

<p>(A) TSC22 inhibits HDM2-mediated p53 ubiquitination. <i>H1299</i> cells were transfected with the indicated plasmids. The transfected cells were treated with MG132 (20 µM) for 5 h before harvest. Cell lysates were immunoprecipitated with an anti-HA antibody. Ubiquitinated p53 was detected by Western blotting with an anti-p53 antibody (DO-1). Ubiquitinated p53 is indicated as Ub(n)-p53 (upper panel). The expression of total p53, HDM2, Flag-TSC-22 and HA-Ub proteins are shown in the lower panels. (B) TSC-22 does not interrupt interaction between p53 and HDM2. <i>H1299</i> cells were transfected with Flag-p53 along with Flag-TSC-22 or a Flag-mock vector in the presence of an HDM2 expression vector. Cell lysates were immunoprecipitated with the anti-p53 (DO-1) antibody followed by Western blotting using the indicated antibodies. Lysate (5%) was analyzed by Western blotting using indicated antibodies (lower panel). (C) TSC-22 inhibits E6-mediated p53 ubiquitination. <i>H1299</i> cells were transfected with the indicated plasmids in presence of an HA-Ub expression vector. 48 h after transfection, the transfected cells were treated with MG132 (20 µM) for 5 h before they were harvested. Cell lysates were immunoprecipitated with an anti-HA antibody. Ubiquitinated p53 was detected by Western blotting with the anti-p53 antibody (DO-1). Ubiquitinated p53 is indicated as Ub(n)-p53 (upper panel). The expression of total p53, Myc-E6, Flag-TSC-22, and HA-Ub proteins are shown in the lower panels. (D) TSC-22 disrupts ubiquitination of p53 in <i>HeLa</i> cells. <i>HeLa</i> cells were cotransfected with Flag-TSC-22 or a Flag-mock vector and HA-Ub expression vector. 48 h after transfection, cells were treated with 20 µM MG132 for 5 h prior to harvesting. Cell lysates were immunoprecipitated with an anti-HA antibody. Ubiquitinated p53 was detected by Western blotting with an anti-p53 antibody (DO-1). (<b>E</b>) Stable TSC-22 knock-down or control <i>HeLa</i> cells were treated with 20 µM MG132 for 5 h prior to harvesting. Ubiquitination of p53 was analyzed as described above.