Fig. 5

SENP1 deSUMOylated and enhanced USP28 deubiquitinating activity towards HIF-1α. a 293T cells were co-transfected with Flag-USP28, His-SUMO2, GFP-SENP1 WT or SENP1 SENP1 CA. SUMO2-conjugated proteins were pulled down by Ni⁺ beads under denaturing conditions and SUMOylated USP28 protein was detected by anti-Flag antibody. b His-SUMO2 HCT116 cells were transfected with control or two shRNAs against SENP1, SUMO2-conjugated proteins were pulled down by Ni⁺ beads under denaturing conditions and subjected to western blot with indicated antibody. c USP28 KD and USP28 WT HCT116 cells were generated by stable expressing of shRNAs against either USP28 or none specific target. These cells were under either normoxia or hypoxia conditions and treated with 20 μM cycloheximide (CHX) for the indicated timepoints. Cells were then lysed and subjected to western blot with indicated antibodies. d USP28 KD or Wild type HCT116 cells were transfected with increased amount of SENP1 plasmid for 36 h, cells were then lysed and subjected to western blot with indicated antibodies