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. Author manuscript; available in PMC: 2017 Jan 24.
Published in final edited form as: Free Radic Res. 2015 Mar 5;49(4):397–410. doi: 10.3109/10715762.2015.1006215

Figure 5.

Figure 5

Effect of disruption of the D5R gene (Drd5) in mice, or silencing of Drd5 in hRPT cells, on PON2 and NOX2 protein and ROS production. (A). Renal PON2 protein is decreased in D5−/− mice relative to their wild-type (D5+/+) littermates. Renal cortical homogenates were immunoblotted with PON2 antibody. Data from D5+/+ mice were given a value of 100%. Data, normalized by β-actin, are shown as mean ± SEM, n = 4–6/group; *P < 0.05 vs D5+/+ mice, t-test. The blot is representative of four to six experiments. (B). Effect of silencing Drd5 on D5R and D1R proteins in hRPT cells. Cells were transfected with human D5R-siRNA (5 nM) or scrambled siRNA (5 nM) for 48 h. Scrambled siRNA was assigned a value of 100%. Data, normalized by β-actin, are shown as mean ± SEM, n = 3–4/group, *P < 0.05 vs scrambled siRNA, t-test. The blots are representative of three to four separate experiments. (C). Effect of silencing Drd5 on PON2 and NOX2 protein expression in hRPT cells. Cells were transfected with human D5R-siRNA (5 nM) or scrambled siRNA (5 nM) for 48 h. Scrambled siRNA was assigned a value of 100%. Data, normalized by β-actin, are shown as mean ± SEM of 3–4/groups, *P < 0.05, t-test vs scrambled siRNA. The blot is representative of three to four separate experiments. (D). Effect of silencing of Drd5 and apocynin on ROS production in hRPT cells. The cells were transfected with D5R-siRNA (5 nM) or scrambled siRNA (5 nM) for 48 h. The cells were then treated with vehicle (Veh) or apocynin (10 µM) for 30 min. ROS production was measured using DCFDA. Vehicle + scrambled siRNA was assigned a value of 100%. Data, normalized by protein concentration, are shown as mean ± SEM, n = 6/groups, *P < 0.05 vs others, one-way factorial ANOVA, Newman–Keuls test. (E). Effect of silencing PON2 on ROS production in hRPT cells. The cells were transfected with PON2-siRNA (10 nM) or scrambled siRNA (10 nM) for 48 h. The cells were then treated with fenoldopam (Fen, 1 µM) or vehicle (Veh) for 24 h. ROS production was measured using DCFDA. Veh + scrambled siRNA was assigned a value of 100%. Data, normalized by protein concentration, are shown as mean ± SEM, n = 6–9/group; *P < 0.05 vs Veh + scrambled siRNA, #P < 0.05 vs others, **P < 0.05 vs fenoldopam + scrambled siRNA, one-way factorial ANOVA, Newman–Keuls test. (F). Effect of silencing PON2 on NOX activity in hRPT cells. Cells were transfected with PON2-siRNA (10 nM) or scrambled siRNA (10 nM) for 48 h, then treated with fenoldopam (Fen, 1 µM) or vehicle (Veh) for 24 h. Membrane NOX activity was measured in the presence of 5 µM lucigenin and 100 µM NADPH. Veh + scrambled siRNA was assigned a value of 100%. Data, normalized by protein concentration, are shown as mean ± SEM, n = 6/group, *P < 0.05 vs Veh + scrambled siRNA, #P < 0.05 vs others, **P < 0.05 vs Fen + scrambled siRNA, one-way factorial ANOVA, Newman–Keuls test.