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. 2015 Mar 15;26(6):1141–1159. doi: 10.1091/mbc.E14-07-1222

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© 2015 Garcia-Alvarez et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 6: — cAMP-induced recruitment of STIM2 and GluA1 to ER-PM contacts. (A–C) TIRF imaging of hippocampal neurons (DIV 6–8) showing cAMP-induced redistribution of YFP-STIM2 to puncta near the PM in the soma (top; see Supplemental Movie S3) and in a proximal dendrite (bottom). (B) cAMP-induced changes in fluorescence intensity in the soma of neurons coexpressing YFP-STIM2 (green, n = 10) and CFP-ER (cyan, n = 10). The vehicle (DMSO) has no effect on YFP-STIM2 intensity (gray, n = 5). (C) Quantification of cAMP-dependent increase in fluorescence intensity in the cell soma for YFP-STIM2 (green, n = 13), ∆SOAR (purple, n = 9), and ∆cyto (dark blue, n = 16). (D–F) TIRF imaging of hippocampal neurons (DIV 6–8) electroporated with mCherry-STIM2 and SEP-GluA1, showing colocalization of mCherry-STIM2 and SEP-GluA1 in puncta both before and after forsk/rolipr addition (see Supplemental Movies S4 and S5). (E) Scatterplots of mCherry-STIM2 against SEP-GluA1 fluorescence intensity (each dot represents a single pixel within the neuron) before and 10 min after forsk/rolipr addition, showing extensive colocalization of mCherry-STIM2 and SEP-GluA1. (F) Pairwise analysis of fluorescence intensity in individual puncta (derived from three neurons) for mCherry-STIM2 and SEP-GluA1 before and 10 min after forsk/rolipr addition. Red bars indicate the median. **p < 0.01, paired t test. (G) TIRF imaging of fixed hippocampal neurons (DIV 7) coexpressing mCherry-STIM2 and GFP-GluA1, treated with forsk/rolipr for 30 min, and stained with an N-terminal GluA1 Ab (Alexa 633) in nonpermeabilizing conditions. Only the mCherry-STIM2 and the surface Ab staining are shown. Arrows point to puncta in the soma (top) and dendritic segment (bottom) that contain both mCherry-STIM2 and surface GluA1. (H) Co-IPs of cortical neurons (DIV 21) treated with DMSO (vehicle) or forsk/rolipr for 30 min with a GluA1 Ab or control IgGs. (I) Dual-color confocal imaging of hippocampal neurons (DIV 21) electroporated with YFP-STIM2 and mCherry, showing cAMP-induced redistribution of YFP-STIM2 (green) in dendritic spines labeled with mCherry (magenta). Snapshots are shown before and 30 min after forsk/rolipr addition. Arrows indicate spines with increased YFP-STIM2 intensity after cAMP elevation (see Supplemental Movie S6). (J–M) Co-IPs of HeLa cells coexpressing mCherry-STIM2 with SEP-GluA1 (J), GFP-cPKA (K), GFP-rPKA (L), and GFP-AKAP (M) treated with forsk/rolipr for 30 min. IPs were carried out with a GluA1 Ab (J), GFP Ab (K–M), or control IgGs. Scale bar, 5 µm.