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. 2013 Jul 4;4(7):e704. doi: 10.1038/cddis.2013.224

Figure 5.

Figure 5

TRIM32 interacts and promotes p73 ubiquitination and degradation, impairing TAp73 transcriptional activity. (a) HEK293 cells were transfected with plasmids encoding myc-TRIM32 and hemagglutinin (HA)-TAp73α, and treated with MG132 (20 μM) for 6 h. Equal amounts of whole-cell extracts were immunoprecipitated with anti-HA (12CA5) antibody, resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted (IB) with anti-myc antibody. The membrane was then stripped and reprobed with anti-HA (Y-11) antibody. (b) HEK293 cells were transfected with plasmid encoding myc-TRIM32 and treated with MG132 (20 μM) for 6 h. Equal amounts of whole-cell extracts were immunoprecipitated with control or anti-p73 (ER-15) antibody, resolved by SDS-PAGE and immunoblotted with anti-myc antibody. The membrane was then stripped and reprobed with anti-p73 (BL906) antibody. (c) HEK293 cells were transfected with plasmids encoding the indicated proteins and treated with MG132 (20 μM) for 6 h. Equal amounts of whole-cell extracts were immunoprecipitated with anti-p73 (ER-15) antibody, resolved by SDS-PAGE and immunoblotted with the indicated antibodies. (d and e) H1299 cells were transfected with constant amounts of plasmid encoding TAp73α, increasing amounts of plasmid encoding TRIM32 (p73/TRIM32 ratio=1 : 1, 1 : 10) and plasmid encoding MDM2 or ΔRING-TRIM32 (ratio=1 : 10). (f) HEK293 cells were transfected with two different small interfering (si)RNA oligonucleotides specific for TRIM32, or control oligonucleotides. Equal amounts of whole-cell extracts were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. Equal amounts of whole-cell extracts were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. (g) Transcriptional analysis with the reporter vector p21Cip1-FL-luc co-transfected with TAp73 alone or together with TRIM32 in a range from 1 : 0.6 to 1 : 2 or TRIM32 alone in HCT116 (left panel) or C17.2 (right panel) cells. Statistical analysis was performed with data from three independent experiments with at least three embryos of each genotype (mean±S.E.M.; **P<0.01)