Figure 3. UCHL1 functions as a deubiquitinating enzyme for HIF-1α.

(a,b) HeLa/5HRE-Luc (a) and HeLa/ODD-Luc (b) cells were transiently transfected with pcDNA4/UCHL1 (UCHL1), pcDNA4/UCHL1 C90S (UCHL1 C90S) or pcDNA4/myc-His A (EV), cultured under normoxic (20%) or hypoxic (1%) conditions for 24 h, and subjected to western blotting using the indicated antibodies and luciferase assay. (c,d) 293T cells transiently transfected with both pcDNA4A/HIF-1α-myc and pMT132 were treated with either scramble-siRNA (Scr) or UCHL1-siRNA (siUCHL1) (c) or additionally transfected with pcDNA4/myc-His A (EV) or pcDNA4/UCHL1 (UCHL1) (d), and cultured with MG132 (30 μg ml⁻¹) for 6 h. Ubiquitinated HIF-1α was immunoprecipitated using an anti-HIF-1α antibody and detected with an anti-ubiquitin antibody (left). One-fiftieth of the whole cell lysate (WCL) was subjected to western blotting with the indicated antibodies (right). (e) Twenty-four hours after the transient transfection with the expression vector for HIF-1α (pcDNA4A/HIF-1α) and either the expression vector for UCHL1 (pcDNA4/UCHL1) or its empty vector, cells were precultured under normoxic (20%) or hypoxic (1%) conditions for 24 h, treated with MG132 (30 μM) under the same oxygen conditions as the preculture, and subjected to immunoprecipitation with the anti-UCHL1 antibody and subsequent western blotting with the indicated antibodies. Paraformaldehyde solution (1%) was used as a cross-linking reagent. One-fiftieth of the WCL was subjected to western blotting with the indicated antibodies. (f) Twenty-four hours after the transient transfection with the expression vectors for HIF-1α (pcDNA4/HIF-1α), VHL-myc (pEF6/VHL-myc), UCHL1 (pcDNA4/UCHL1) or their empty vector, cells were treated with MG132 (30 μg ml⁻¹) for an additional 6 h. VHL-myc was immunoprecipitated using an anti-myc antibody and co-precipitated HIF-1α was detected. One-fiftieth of the WCL was subjected to western blotting with the indicated antibodies (lower). (g,h) RCC4 plus vector alone and RCC4 plus VHL cells were transiently transfected with p5HREp-luc and either pcDNA4/UCHL1 (UCHL1) or pcDNA4/myc-His A (EV), cultured under normoxic (20%) or hypoxic (0.1%) conditions for 24 h, and subjected to western blotting using the indicated antibodies and luciferase assay. Mean±s.d. n=3. *P<0.05; NS, not significant, Student’s t-test (g) and Dunnett’s test (a,b). IP, immunoprecipitation; IB, immunoblot; WB, western blotting.