Fig. 1

Hypoxia enhanced the deubiquitinating activity of USP28 on HIF-1α. a HCT116 and A549 cells were cultured under normoxic (16% O₂) or hypoxic (5% O₂) conditions for 24 h. Cells were then lysed and USP28 protein levels were analyzed by western blot. The number on the bottom indicates signal intensity of USP28 protein against GAPDH. b 293T cells were transfected with Flag-tagged USP28 for 24 h and then cultured under normoxic or hypoxic conditions for additional 24 h. Then, Flag M2 beads were used to purify USP28 proteins in 293T cells. HA-HIF-1α, Myc-FBXW7 and His-Ubiquitin were co-transfected in 293T cells for 36 h. MG132 was added 4 h before cells were harvested. Ubiquitinated HIF-1α was purified from 293T cells using anti-HA antibody and used as the substrate of USP28 in vitro. BSA, normoxic USP28 or hypoxic USP28 proteins were added to ubiquitinated HIF-1α-FBXW7 complex for 1 h at 30 °C. 2Χ SDS loading buffer was then added to terminate the enzymatic reaction and the materials were subjected to western blot with indicated antibodies