Fig. 1. Ca²⁺-dependent, biphasic activation of PLC in insulin-secreting cells. (A) MIN6 cells were transiently transfected with PH_PLC-GFP and visualized 24 hours later with evanescent wave microscopy under basal conditions and after stimulation with 100 µM carbachol at the time points i and ii indicated in B. Bars 5 µm. (B) Time-course of PH_PLC-GFP translocation in the cell shown in A after stimulation with 100 µM carbachol. (C) Time-course of PH_PLC-GFP translocation in response to 100 µM carbachol in Ca²⁺-deficient medium containing 2 mM EGTA. (D) Effect of Ca²⁺ removal with addition of 2 mM EGTA on the steady-state PH_PLC-GFP fluorescence during stimulation with 100 µM carbachol. (E) Time-course of PH_PLC-GFP translocation in response to 100 µM carbachol in the presence of 0.5 mM La³⁺. The inset shows the entire experiment, including the rise of PH_PLC-GFP fluorescence that occurs upon addition of La³⁺. The fluorescence was normalized against the level prior to carbachol stimulation. (F) Mean±s.e.m. for the effects of Ca²⁺ removal and La³⁺ addition on PH_PLC-GFP fluorescence. The peak amplitude was defined as the maximal change from initial fluorescence and the plateau amplitude was calculated from the off-response upon washout of carbachol. ^**P<0.001; ^*P<0.01.