Fig. 3

NEDD4L regulates UBE2T protein level by promoting its ubiquitylation. A Overexpression of NEDD4L decreased the protein level of UBE2T in a dose-dependent manner. H1299 cells were transfected with increasing amounts of HA-NEDD4L, followed by IB with indicated Abs. B Blockade of proteasome-mediated degradation rescued NEDD4L-mediating UBE2T down-regulation. H1299 cells were transfected with different doses of HA-NEDD4L (0, 0.5, 1 and 2 μg) for 48 h. The proteasome inhibitor, MG-132, was then added to cells and MG-132 co-treatment was allowed to occur for 8 h, followed by IB. C NEDD4L shortened the half-life of exogenous UBE2T protein. After transfection with relevant plasmids for 48 h, 293 cells were switched to fresh medium (10% FBS) containing cycloheximide (CHX) and incubated for indicated time periods before being harvested for IB. The band density was quantified using ImageJ software and plotted. D NEDD4L RNAi silencing extended protein half-life of endogenous UBE2T. H358 cells were transfected with either control RNAi, or Si-NEDD4L for 48 h. Cells were cultured in fresh medium containing CHX and incubated for indicated time periods before being harvested for IB. The band density was quantified using ImageJ software and plotted. E NEDD4L promoted UBE2T ubiquitylation in vivo: 293 cells were transfected with indicated plasmids, lysed under denatured condition at 6 M guanidinium solution, followed by Ni-beads pull-down. Washed beads were boiled for IB to detect polyubiquitylation of exogenous UBE2T