Figure 6. GLP-1-stimulated ERK activation in MIN6 cells is mediated via local Ca²⁺ signalling.

MIN6 cells incubated in KRB plus 1 mM glucose were loaded with 100 µM EGTA-AM or BAPTA-AM at room temperature prior to treatment with 10 nM GLP-1 plus 16.7 mM glucose for the times indicated. a) Proteins were resolved by SDS-PAGE and Western blotted using anti-phospho-ERK1/2 (pERK) and anti-ERK2 (ERK2) antibodies. A representative blot is shown with densitometric analysis of the results below showing mean +S.E.M. (n = 3). Data were analysed by two-way ANOVA with Bonferroni's multiple comparison test compared to GLP-1 plus glucose at each time point; *, P<0.05; **, P<0.01; ***, P<0.001. b) MIN6 cells were treated as in a) but in addition, loaded with 2 µM fura-2-AM and [Ca²⁺]_i levels measured by epifluorescence microscopy. i) The mean increase in [Ca²⁺]_i represented as area under the curve (A.U.C.) during ii) 10 min or iii) 30 min stimulation, mean +S.E.M. (n>30). Statistical comparisons were by one-way ANOVA with Dunnett's range test compared to GLP-1 plus glucose in the absence of chelator. ***, P<0.001.