Figure 3: PPARγ functions as an E3 ligase to induce the ubiquitination of p65.

(a) HEK293T cells were transiently transfected with PPARγ for 36 h. Cell lysates were subjected to denatured immunoprecipitation and western blotting. Cells were treated with 10 μM MG132 for 6 h before cell lysis. (b) HT29 cells were transfected with scrambled shRNA or PPARγ shRNA for 36 h. Cell lysates were then subjected to denatured immunoprecipitation western blotting. Cells were treated with 10 μM MG132 for 6 h before cell lysis. (c) PPARγ^−/− MEF cells or PPARγ^−/− MEF cells were transiently transfected with PPARγ, C139A or C193A separately. Cell lysates were subjected to denatured immunoprecipitation and western blotting. Cells were treated with 10 μM MG132 for 6 h before cell lysis. (d) In vitro polyubiquitin formation analysis was performed (see experimental procedures) in reaction buffer contained different E2 (UBCH3, UBCH5a, b, c) with 10ng PPARγ or (e) containing UBCH3 with 10 ng PPARγ, C139A or C193A as indicated. Reactions were incubated at 30 °C for 2 h. The ubiquitinated products were detected with an ubiquitin antibody. (f) GST pull-down assay in which GST-PPARγ or GST alone bound to glutathione-agarose beads was incubated with recombinant p65. (g) In vitro ubiquitination of p65 analysis was performed (see experimental procedures) in the reaction buffer contained UBCH3, p65 (10 μg) and 10 ng PPARγ (WT or C139A) as indicated. Reactions were incubated at 30 °C for 2 h, resolved by SDS–PAGE and the ubiquitinated products were detected with p65 antibody. (h) HEK293 cells were transfected with PPARγ, his-p65 or K28R. Cell lysates were subjected to denatured Ni-NTA pull-down and western blotted. Cells were treated with 10 μM MG132 for 6 h before cell lysis. (i) HT29 cells were transfected with his-p65 or his-p65/K28R plasmids and after 36 h, cells were treated with cycloheximide (30 μg ml⁻¹) for 0, 0.5, 1, 2 and 5 h to inhibit de novo protein synthesis and harvested for p65 by western blot. Data are triplicates from three independent experiments.