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. 2018 Jan 5;32(3):1170–1183. doi: 10.1096/fj.201700834R

Figure 6.

Figure 6.

pH-dependent activation of GPR68 acts via cAMP/PKA/CREB to increase IL-6 production by pancreatic CAFs. A) CAFs transfected with control or GPR68 siRNA were cultured in pH medium for 30 min, treated with 1 mM isobutylmethylxanthine for 10 min and cellular cAMP was measured by the HitHunter cAMP assay. Data are means ± sem, n = 3; 2-way ANOVA followed by Sidak post hoc test. *P < 0.05, ****P < 0.0001. B) Real-time qPCR analysis of GPR68 and IL-6 expression in CAFs cultured for 6 h in pH 7.4, pH 6.8, or pH 6.8 medium in the presence of the PKA inhibitors, H89 and PKI. Results were normalized to those at pH 7.4. Data are means ± sem, n ≥3; 1-way ANOVA, followed by Tukey post hoc test. ****P < 0.001(ns, nonsignificant). C) Immunoblot of CREB and Ser131 phosphorylated CREB (p-CREB) of CAFs cultured in pH medium (pH 7.4–6.4) for 6 h. Activation of CREB is shown as p-CREB/CREB. Data are means ± sem, n = 3; 1-way ANOVA, followed by Dunnett post hoc test. **P < 0.01, ***P < 0.001. D) Real-time qPCR analysis of GPR68 and IL-6 expression in CAFs cultured for 6 h in pH 7.4, pH 6.8, or pH 6.8 medium in the presence of the CREB inhibitors, naphthol AS-E and 666-15. Results are normalized to those at pH 7.4. Data are means ± sem, n = 3; 1-way ANOVA, followed by Tukey post hoc test. ***P < 0.001, ****P < 0.0001(ns, nonsignificant).