FIGURE 6.

NOX4-dependent H₂O₂ production in shCANX-treated NOX4-HEK293 cells. A and E, normalized, relative mRNA level for CANX and NOX4 in controls (scrambled shRNA: shSCR, shGFP) and shCANX-treated NOX4-HEK293 cells (sh1 and sh2). B and F, representative Western blot with densitometry for NOX4 and CANX protein expression normalized to β-Actin and shSCR in controls (shSCR and shGFP) and after shRNA-mediated CANX knockdown (sh1 and sh2). C, relative diphenylene iodonium-sensitive (10 μm) Amplex Red/HRP assay of controls (shSCR and shGFP) and after shRNA-mediated CANX knockdown (sh1 and sh2) normalized to protein amount (μg). D, relative luminol/HRP assay in controls (shSCR and shGFP) and after shRNA-mediated CANX knockdown (sh1 and sh2). 100,000 cells per measurement were used and CU (chemiluminescence unit) was normalized to protein amount (μg) and shSCR control. For all ROS measurements, three independent measurements were performed for each of the three biological replicates. Also shown are representative Western blots for Na/K-ATPase (B) and HSPA5 (F) protein expression in controls (shSCR and shGFP) and after shRNA-mediated CANX knockdown (sh1 and sh2), n ≥ 3, mean ± S.E. *, p < 0.05; **, p < 0.01; ***, p < 0.001 relative to the corresponding shSCR-treated cells.