A sustained global increase in [Ca²⁺]_i is insufficient for the prolonged activation of ERK.

<p>(a and b) MIN6 cells were preincubated for 1 h in KRB supplemented with 1 mM glucose. Cells were incubated in 50 mM K⁺ (K50) in the presence or absence of 10 µM nifedipine for the times indicated (All statistical comparisons were by one-way ANOVA with Bonferroni's multiple comparison test compared to K50 at each time point; *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001). a) Proteins were resolved by SDS-PAGE and Western blotted using anti-phospho-ERK1/2 and anti-ERK2 antibodies. A representative blot is shown above densitometric analysis of the results showing mean +S.E.M. (n = 3). b) In cells loaded with 2 µM fluo-4-AM, fluorescence (as an index of [Ca²⁺]_i) was measured using a NOVOstar platereader. (c and d) MIN6 cells were preincubated for 1 h in KRB supplemented with 1 mM glucose. Cells were then treated with 10 µM ionomycin or 50 mM K⁺ (K50) for the times indicated. c) In cells loaded with 2 µM fluo-4-AM, fluorescence (as an index of [Ca²⁺]_i) was measured using a NOVOstar platereader. ***, <i>P</i><0.001 for K50 versus ionomycin at equivalent time points. d) After treatments, proteins were resolved by SDS-PAGE and Western blotted using anti-phospho-ERK1/2 (pERK) or anti-ERK2 (ERK2) antibodies. A representative blot is shown with densitometric analysis of the results below showing mean +S.E.M. (n = 3).