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. 2015 Mar 13;28(10):1267–1276. doi: 10.1093/ajh/hpv018

Figure 4.

Figure 4.

Effect of MAPK inhibitor on D3R and AT2R agonist-mediated effects on phospho-ERK, Na+-K+-ATPase activity, and D3R and AT2R colocalization and coimmunoprecipitation in renal proximal tubule cells (RPTCs). (a) Effect of D3R and AT2R agonists on phospho-ERK1/2 (p-ERK1/2) protein expression in RPTCs. RPTCs were treated with vehicle, and the D3R agonist, PD128907 (10−8 M), and AT2R agonist CGP42112A (10−10 M), or their combination. The cell lysates were immunoblotted with anti-phospho-ERK antibody and anti-total (t) ERK antibody. P-ERK expression was corrected by t-ERK protein expression. Data are expressed as mean ± SEM. *P < 0.05 vs. Vehicle, # P < 0.05 vs. others, n = 5 (1-way analysis of variance [ANOVA], Holm-Sidak test). (b) Effect of D3R and AT2R agonists on the Na+-K+-ATPase activity of RPTCs in the absence or presence of MAPK inhibitor PD98059. RPTCs were pretreated with vehicle or PD98059 (10−5 M) for 10 minutes, then treated with PD128907 (10−8 M) in combination with CGP42112A (10−10 M) for 15 minutes. Protein concentration was used to normalize the results. Data are expressed as mean ± SEM. *P < 0.05 vs. Vehicle, # P < 0.05 vs. others, n = 6 (1-way ANOVA, Holm-Sidak test). (c) Effect of D3R and AT2R agonists on the cellular distribution and colocalization of D3R and AT2R in RPTCs in the absence and presence of MAPK inhibitor PD98059. RPTCs were pretreated with PD98059 (10−5 M) for 10 minutes, then treated with PD128907 (10−8 M) in combination with CGP42112A (CGP, 10−10 M) for 15 minutes. Colocalization of D3R (red) and AT2R (green) was observed as discrete yellow areas in merged images. Images are representative of 3 experiments using different cell preparations. (d) Effect of D3R and AT2R agonists on the coimmunoprecipitation of D3R and AT2R in RPTCs in the absence and presence of MAPK inhibitor PD98059. RPTCs were pretreated with vehicle or PD98059 (10−5 M) for 10 minutes, then treated with PD128907 (10−8 M) in combination with CGP42112A (CGP, 10−10 M) for 15 minutes. Data are expressed as mean ± SEM. *P < 0.05 vs. Vehicle, # P < 0.05 vs. others, n = 6 (1-way ANOVA, Holm-Sidak test).