Fig. 4: Human GLP-1R–SPHKAP association via shared VAPB binding and SPHKAP-dependent GLP-1R-induced VAPB-localised PKA activity.

a SPHKAP-EGFP co-IP with SNAP/FLAG-hGLP-1R in Control versus VAPB RNAi-treated INS-1 832/3 SNAP/FLAG-hGLP-1R cells under vehicle (Veh) versus exendin-4 (Ex-4)-stimulated conditions; blots representative of n = 5 biologically independent experiments; GFP-positive fragments detected due to intrinsic instability of co-IPed full-length fusion protein. b Quantification of SPHKAP-EGFP over SNAP/FLAG-hGLP-1R levels from (a); n = 5 biologically independent experiments, p values as indicated by one-way ANOVA with Šidák post-hoc test. c Confocal microscopy analysis of SPHKAP-EGFP versus mitochondria (imaged with MitoTracker Red) localisation in INS-1 832/3 cells treated with Control versus VAPB RNAi; size bars, 5 μm; images representative of n = 3 biologically independent experiments. d Confocal microscopy analysis of SPHKAP-EGFP versus ER (imaged with ER-Tracker Red) localisation in INS-1 832/3 cells; size bar, 10 μm; images representative of n = 3 biologically independent experiments. e SPHKAP-EGFP co-localisation (Mander’s coefficient) with mitochondria (Mito) versus ER; n = 5 cells from 3 independent experiments, p value as indicated by two-tailed unpaired t-test. f Ex-4-stimulated cAMP responses in Control versus SPHKAP RNAi-treated INS-1 832/3 cells, measured with cADDis cAMP biosensor; left, cAMP traces; right, AUC quantification; n = 3 biologically independent experiments; p value as indicated by two-tailed paired t-test. g Ex-4-stimulated global PKA responses in Control versus SPHKAP RNAi-treated INS-1 832/3 cells; left, PKA traces; right, AUC quantification; n = 4 biologically independent experiments; p value as indicated by two-tailed paired t-test. h Ex-4-stimulated mitochondrial PKA responses in Control versus SPHKAP RNAi-treated INS-1 832/3 cells; left, PKA traces; right, AUC quantification; n = 4 biologically independent experiments; p value as indicated by two-tailed paired t-test. i, VAPB-localised PKA activity (measured by FluoSTEP) in response to 100 nM Ex-4 stimulation in Control versus SPHKAP RNAi-treated INS-1 832/3 cells expressing pcDNA3-GFP11(x7)-VAPB + pcDNA3.1(+)-FluoSTEP-AKAR; maximal responses (to FSK + IBMX) shown; kinetic traces (left) and corresponding AUCs for the agonist-stimulated period (right) shown; n = 5 biologically independent experiments; p value as indicated by two-tailed paired t-test. Data are mean ± SEM.