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. Author manuscript; available in PMC: 2022 Sep 8.
Published in final edited form as: Hypertension. 2021 Sep 8;78(4):1027–1038. doi: 10.1161/HYPERTENSIONAHA.121.16981

Figure 5.

Figure 5.

Regulation of phosphorylation of NCC by sPRR in Flp-In T-REX 293 NCC cell line. (A) Validation of siRNA-mediated PRR knockdown and its effect on pNCC-T53 expression in Flp-In T-REX 293 NCC cell line. The cells were transfected with PRR siRNA and then treated with 100 nM prorenin for 24 h, then NCC and pNCC-T53 protein expression was analyzed by immunoblotting and densitometric analysis. PRR-probed membrane was stripped and re-probed with anti-β-actin antibody as an internal control for equal loading of the samples. N = 6 per group. Data are mean ± SEM. *p<0.05, **p<0.01, ***p<0.001 vs. Control; #p<0.05 vs. Prorenin (ANOVA with the Bonferroni test). (B) Effect of sPRR and PF429242 on expression of abundance of NCC and pNCC-T53. Flp-In T-REX 293 NCC cells were treated with 10 nM sPRR-His, or 5 μM PF429242, or 5 μM PF429242 in combination with 10 nM sPRR-His for 24 h, and the abundnaces of PRR, NCC, and pNCC-T53 were analyzed by immunoblotting and densitometric analysis. PRR-probed membrane was stripped and re-probed with anti-β-actin antibody. N = 6 per group. Data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, vs. Control; ##p < 0.01 vs. PF; &p < 0.05 vs. sPRR-His (ANOVA with the Bonferroni test). (C, D) sPRR-His/AT1R signal regulates NCC-T53 phosphorylation. Cells were pretreated with 10 mM losartan (C) or transfected with AT1R siRNA (D), and then treated with 10 nM sPRR-His for 24 h. PRR, NCC, and pNCC-T53 protein expression was analyzed by immunoblotting and densitometric analysis. PRR-probed membrane was stripped and re-probed with β-actin as an internal control for equal loading of the samples. N = 6 per group. Data are mean ± SEM. *p<0.05, **p<0.01 vs. Control; &&p<0.01, &&&p<0.001 vs. sPRR-His (ANOVA with the Bonferroni test). (E) Effect of mineralocorticoid receptor antagonist on the level of pNCC-T53. Cells were pretreated with 10 μM Eplerenone, and then treated with 10 nM sPRR-His for 24 h, then NCC and pNCC-T53 protein abundance was analyzed by immunoblotting and densitometric analysis. pNCC-T53-probed membrane was stripped and re-probed with anti-β-actin antibody. N = 3 per group. Data are mean ± SEM. ***p < 0.001, vs. Control (ANOVA with the Bonferroni test). (F) sPRR blocked sodium uptake via the inhibition of NCC in Flp-In T-REX 293 NCC cells. The values were normalized by protein content. N = 6 per group. Data are mean ± SEM. Statistical analysis was performed by using ANOVA with the Bonferroni test.