Figure 3. NOX-dependent ROS generation is required for Src activation and EGFR phosphorylation by PTHrP (1–34).

(A) MCs were incubated with DCFH-DA at 37°C for 30 min and then treated or untreated with selective NOX inhibitor, apocynin, before 100 nM PTHrP (1–34) stimulation for 5 min, and H₂O₂ production was assayed by flow cytometry (*P<0.05 vs control, n=3). (B) MCs were incubated with DHE at 37°C for 30 min and then treated with apocynin or NAC before 100 nM PTHrP (1–34) stimulation for 5 min. Superoxide production was assayed by flow cytometry (*P<0.05 vs control, n=3). (C) MCs were treated or untreated with apocynin or NAC before 100 nM PTHrP (1–34) stimulation for 5 min, and NOX activity was assayed by measuring the NADPH-dependent superoxide dismutase (SOD)-inhibitable cytochrome c reduction (*P<0.05 vs control, n=3). (D) MCs were pretreated with NAC or apocynin before PTHrP (1–34) treatment for 1 h. Src phosphorylation at Y416 and EGFR phosphorylation at Y845 were assessed by western blot (*P<0.05 vs control, n=4).