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. Author manuscript; available in PMC: 2021 Aug 18.
Published in final edited form as: Hypertension. 2021 Jul 19;78(3):816–830. doi: 10.1161/HYPERTENSIONAHA.120.15423

Figure 3. Deletion of GPR35 (G-protein–coupled receptor 35) increased eNOS (endothelial nitric oxide synthase) activation in human aortic endothelial cells (HAECs).

Figure 3.

The protein levels of total eNOS (noted as eNOS), phosphorylated eNOS at Ser1177(p-eNOSser1177), and β-actin were determined by Western blot analysis using β-actin as a housekeeping protein. The extent of eNOS phosphorylation was calculated as the ratio of p-eNOS (Ser1177) to total eNOS (p-eNOSser1177/eNOS). Representative images are shown beneath the quantifications. A, The eNOS, p-eNOSser1177, and p-eNOSser1177/eNOS in GPR35WT and GPR35KO mouse aortic ECs (MAECs). n=7 in GPR35WT, n=8 in GPR35KO ECs. Mann-Whitney U tests were used. *P<0.05 vs GPR35WT. B, The eNOS, p-eNOSser1177, and p-eNOSser1177/eNOS in HAECs infected with Ad-sh-GPR35 or Ad-β-gal. n=6 per group. Mann-Whitney U tests were used. *P<0.05 vs Ad-Ad-β-gal. C, The eNOS, p-eNOSser1177, and p-eNOSser1177/eNOS in HAECs infected with Ad-sh-GPR35 or Ad-β-gal. n=6 per group. Mann-Whitney U tests were used. *P<0.05 vs Ad-Ad-β-gal. D, Functional assays including migration (left) and proliferation (right) in HAECs transfected with siRNA against eNOS (si-eNOS) and siRNA against GPR35 (si-GPR35), using scramble oligo as control. Representative images are shown next to the quantifications. Scale bar, 200 μm. n=5 per group in the migration assay, n=4 per group in the proliferation assay. Kruskal-Wallis tests were done, followed by Mann-Whitney U tests with Hommel’s adjustment. *P<0.05 between the 2 groups in post hoc comparisons. E, Quantification of fluorescent staining of NO production in HAECs transfected with siRNA against GPR35 (si-GPR35), using scramble oligo as control. Representative images are shown. Scale bar, 200 μm. n=6 per group. Mann-Whitney U tests were used. *P<0.05 vs Scramble. F, Quantification of fluorescent staining of reactive oxygen species (ROS) in HAECs transfected with si-GPR35, using scramble oligo as control. Representative images are shown. Scale bar, 200 μm. n=6 per group. Mann-Whitney U tests were used. *P<0.05 vs Scramble. In all the figures, horizontal lines show mean±SD.