Figure 4.

Ablation of BVR protein prevents ISO-induced HO-2 protein expression. A) Cardiomyocytes were infected with retrovirus expressing siBVR, 6 h later, cells were treated with 10 μM ISO. Samples were collected at 24, 48, 72, and 96 h posttreatment. Cell lysates were analyzed by gel electrophoresis and Western blot analysis. Blots were probed with antibodies against rat BVR and HO-2. β-Actin served as a control. B) Cardiomyocytes were infected with a retrovirus expressing siHO-2. Expression of BVR and HO-2 was measured as in A. C) BVR expression regulates the stability of the HO-2 protein. In the first experiment, HEK293A cells were cotransfected with pcDNA-hBVR and pcDNA-hHO-2, then stimulated with ISO for 24 h, followed by treatment with the protein synthesis inhibitor, cycloheximide (10 μg/ml), for the indicated intervals. Expression of hHO-2 and hBVR in HEK293A cells was measured by Western blot. In the second series, the cells were transfected with pcDNA-hHO-2 and treated with retrovirus expressing sihBVR. These data were replicated in ≥2 independent experiments. D) Cardiomyocytes were treated with 10 μM ISO, infected with siBVR virus, or treated with the proteasome inhibitor MG132 (50 μg/ml). HO-2 and BVR protein expression were detected by Western blot. β-Actin served as a control.