Fig 1: Ca2+-dependent ERK activation through Ras and RasGRF2 in MIN6 cells.(A) Left: Representative single-cell timecourse of RaichuEV-Ras emission ratio (Ras, blue curve) and RCaMP1d fluorescence intensity (Ca2+, red curve) in MIN6 cells sequentially treated with TEA and CaCl2. Curves representative of n = 43 cells from 3 independent experiments. Additional representative traces shown in Fig. S4. Right: Quantification of maximum TEA-stimulated RaichuEV-Ras emission ratio change (ΔR/R) in MIN6 cells before (−) and after (+) CaCl2 addition. n = 43 cells from 3 independent experiments. Data analyzed using paired, two-tailed Student’s t-test. (B) Right: Representative average timecourse of EKAREV emission ratio in TEA-stimulated MIN6 cells co-expressing mCherry-NLS (control, dark blue curve; n = 18 cells) or dominant-negative HRasN17 (black curve). n = 18 (control) and 12 (HRasN17) cells, representative of 5 and 7 independent experiments, respectively. Left: Quantification of maximum emission ratio change (ΔR/R; upper) or time to half-maximal response (T1/2; lower) in TEA-stimulated MIN6 cells expressing EKAREV plus either HRasN17 (+) or mCherry-NLS (−). n = 59 (+) and 65 (−) cells from 5 and 7 independent experiments, respectively. Data analyzed using Mann-Whitney U-test. Solid and dashed lines in violin plots show median and quartiles, respectively. (C) Representative western blot (left) and quantification of RasGRF2 expression (upper right; n = 4 independent experiments) and ratio of phosphorylated ERK to total ERK expression (pERK/tERK; lower right; n = 3 independent experiments) in MIN6 cells expressing either control shRNA (shCon) or shRNA targeting RasGRF2 (shGRF2). Data analyzed using unpaired, two-tailed Student’s t-test. Bars show mean ± s.e.m (normalized to shCon).