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. 2010 Oct 5;285(50):38832–38840. doi: 10.1074/jbc.M110.124958

FIGURE 7.

FIGURE 7.

Effects of ROS on Cav-1 mRNA expression and protein degradation. A, H460 cells were either left untreated or treated with DMNQ (5 μm), H2O2 (100 μm), or FeSO4 (50 μm) for 24 h. Cav-1 and GAPDH mRNA expressions were then determined by quantitative real time PCR. The relative mRNA expression was determined by using the comparative CT method as described under “Materials and Methods.” B and C, H460 cells were pretreated with proteasome inhibitor lactacystin (LAC) (10 μm), MG132 (MG) (25 μm), or with lysosome inhibitor concanamycin A (CMA) (1 μm) for 1 h and then treated with DMNQ (5 μm) or H2O2 (100 μm) for 24 h. Cav-1 expression was determined by Western blots using anti-Cav-1 antibody. Con, control. D and E, cells were treated with DMNQ (5 μm) in the presence or absence of MnTBAP (50 μm) or with H2O2 (100 μm) in the presence or absence of CAT (7,500 units/ml). Cell lysates were immunoprecipitated with anti-Cav-1 antibody, and the immune complexes were analyzed for ubiquitin (Ub) by Western blots using anti-ubiquitin antibody. Analysis of ubiquitin was performed at 2 h post-treatment where ubiquitination was found to be maximal. Immunoblot signals were quantified by densitometry, and mean data from three independent experiments (one of which is shown here) was normalized to the result obtained in control cells. Data are the mean ± S.D. (n = 3). *, p < 0.05 versus nontreated control; #, p < 0.05 versus treated control.