Fig. 7

TRIM3 facilitated P53 K48-linked poly-ubiquitination and degradation. a In the presence of the proteasome inhibitor MG132, the degradation effect of TRIM3 on P53 did not further increase P53 protein levels. MCF-7 cells were transfected with with siControl or siTRIM3. After 24 h, cells were treated with 20 uM MG132/vehicle for 7 h. Cell lysates were prepared for Western blot analysis. The results are representative for three independent experiments. b, c TRIM3 decreased P53 half-life in MCF-7 cells. MCF-7 cells were transfected with with siControl or siTRIM3. After 24 h, cells were treated with 100 µM cycloheximide/vehicle for indicated times. Cell lysates were prepared for Western blot analysis. The results are representative for three independent experiments. The P53 relative density was measured by Image J software. d TRIM3 increased the overall poly-ubiquitination of P53. HEK293 cells were transfected with 1 µg GFP-P53 together with 0.5 µg Myc-TRIM3 or Myc vector. After 24 h, cells were transfected with 1 µg HA-Ub plasmid. After another 24 h, the cell extracts were immunoprecipitated with HA antibody. The poly-ubiquitinated P53 was detected via western blotting analysis. e TRIM3 increases K48-linked poly-ubiquitination of P53. HEK293 cells were transfected with 1 µg GFP-P53 together with 0.5 µg Myc-TRIM3 or Myc vector. After 24 h, cells were transfected with 1 µg HA-K48-Ubi plasmid. After another 24 h, the cell extracts were immunoprecipitated with HA antibody. The K48-linked poly-ubiquitinated P53 was detected via western blotting analysis