Supplementary Figure S2.

(a) One representative western blot among the ones considered in the densitometric analysis reported in Figure 1b. The cell lysates were probed with the indicated antibodies to show variations in pERK/ERK ratio. (b) Western blot analysis of HEK DKO Gαq/11; CasR; NFAT-Luc cells nontransfected or transfected with GNAQWT, GNAQR183Q, GNA11^WT, or GNA11^R183C pcDNA3.1(+)-N-HA plasmids. The cell lysates were probed with the indicated antibodies to show equal expression of the transgenes. (c) RT-qPCR analysis of CaSR expression levels in TIME GNAQ WT or GNAQ p.(R183Q) cell lines and in a CD31⁺ cell population isolated from a SWS vascular lesion. HEK DKO;CaSR were used as positive control of the TaqMan expression assay. Mean ± SD of technical quadruplicate of one experiment representative of two independent experiments. (d) TIME parental cells were loaded with intracellular calcium probe Fluo-8 and treated for 20 minutes with vehicle or 1 μM CM4620. After treatment, cells were stimulated with thrombin 1U/ml and fluorescence recorded for 300 seconds. The graphs represent an average of four independent experiments. ERK, extracellular signal–regulated kinase; HEK, human embryonic kidney; pERK, phosphorylated ERK; TIME, telomerase-immortalised microvascular endothelial; WT, wild-type.