Fig. 3: RBX1 negatively regulates UBE2F protein levels.
A, B UBE2F protein levels are decreased by RBX1 and increased by RBX2. H1299 cells were transfected with siRNAs targeting RBX1/2 or indicated plasmids. Protein levels of UBE2F were determined by western blot and normalized against β-actin. C, D RBX1 and RBX2 have little or no effect on the mRNA levels of UBE2F. H1299 cells were transfected with siRNAs targeting RBX1/2 or indicated plasmids. mRNA levels of UBE2F, RBX1, and RBX2 were determined by RT-PCR and normalized against β-actin. E Platinum treatment impairs the protein interaction between UBE2F and RBX1. H1299 cells were transfected with indicated plasmids and then treated with cisplatin (Cis) or carboplatin (Carbo) for 48 h. UBE2F proteins were purified by Flag beads, and their protein interactions with endogenous RBX1, RBX2, and NOXA were determined by western blot. F Platinum treatment inhibits RBX1-mediated UBE2F poly-ubiquitylation. H1299 cells were co-transfected with indicated plasmids and then treated with cisplatin (Cis) or carboplatin (Carbo) for 48 h. UBE2F proteins were purified by Flag beads, and poly-ubiquitylation of UBE2F was determined by western blot. G Downregulation of RBX1 extends the half-life of UBE2F. A549 cells were transfected with control or RBX1 siRNA for 72 h and then treated with 50 μg/mL CHX at the indicated time. The half-life of UBE2F was calculated by mean value from three independent experiments. Shown are average values with standard deviation (s.d.). **P < 0.001 for the indicated comparison. H UBE2F exerts a negative correlation with RBX1 in lung adenocarcinoma. 20 lung adenocarcinoma tissues were lysed. The UBE2F and RBX1 protein levels were compared against β-actin by western blot.
