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. Author manuscript; available in PMC: 2022 May 5.
Published in final edited form as: J Vasc Res. 2021 May 5;58(5):277–285. doi: 10.1159/000516044

Figure 3. Inhibitors against Rap1, B-Raf, and Mek1/2 eliminate elevated Pi-induced ERK1/2 phosphorylation.

Figure 3.

Elevated Pi -induced ERK1/2 phosphorylation was assessed with small molecule inhibitors against Rap1 (GGTI298), B-Raf (GDC0879), and Mek1/2 (U0126). A) Schematic represents the effects of each small molecule inhibitor on proteins involved in the signaling pathway. B) Primary murine VSMCs were incubated in Pi-free DMEM with varying inhibitor concentrations for 16 hours starting at 20μM GGTI298, 10μM GDC0879, or 20μM U0126 and diluted serially with a dilution factor of 10, then induced with 3.0mM Pi (or given controls), and phosphorylated ERK1/2 was quantified by western blot. C) VSMCs were incubated in Pi-free DMEM overnight, then Pi-free DMEM with varying concentrations of inhibitors (20μM GGTI298, 10μM GDC0879, or 20μM U0126) for 1 hour, and induced with either 1.0mM or 3.0mM Pi, then ERK1/2 was visualized by western blot. D) Induction of P-ERK1/2 after pre-incubation with inhibitors and induction with 1.0mM Pi or 3.0mM Pi was quantified by In-Cell ELISA assay. (two-way ANOVA post-hoc Sidak in D: ns = p > 0.05, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001)