Figure 2: UCHL1 increases the stability of HIF-1α.
From: UCHL1 provides diagnostic and antimetastatic strategies due to its deubiquitinating effect on HIF-1α
(a,b) HeLa (a) and 293T (b) cells were transiently transfected with either pcDNA4/UCHL1 (UCHL1) or pcDNA4/myc-His A (EV) (a) or treated with either scramble-siRNA (Scr) or UCHL1-siRNA (siUCHL1) (b), cultured under normoxic (20%) or hypoxic (0.1%) conditions for 24 h, and then subjected to western blotting using the indicated antibodies. The intensity of the bands in the left panel of b was quantified and relative HIF-1α protein levels after the siUCHL1-1 or -2 treatment to those after the Scr-siRNA treatment were shown in the right panel of b. (c,d) HeLa/ODD-Luc (c: left), MCF7/ODD-Luc (c: right), 293T (d: left) and MDA-MB-436 (d: right) were transiently transfected with either pcDNA4/UCHL1 (UCHL1) or pcDNA4/myc-His A (EV) (c), or treated with either scramble-siRNA (Scr) or UCHL1-siRNA (siUCHL1) (d), cultured under normoxic (20%) or hypoxic (1%) conditions for 24 h, and then subjected to western blotting using the indicated antibodies and the luciferase assay. (e) HeLa cells were transiently transfected with pcDNA4/UCHL1 (UCHL1) or pcDNA4/myc-His A (EV), cultured under hypoxic conditions (0.1%) for 24 h, treated with cycloheximide (10 μg ml−1) under normoxic conditions (20%) for the indicated period, and harvested for western blotting with the indicated antibodies. (f) Western blotting of the HeLa cell lysate after the hypoxic treatment (1%) for the indicated periods with or without the transient transfection of the UCHL1 expression vector. Mean±s.d. n=3. *P<0.05, **P<0.01, Student’s t-test (b,c) and Dunnett’s test (d).
