Fig. 1: Immunogold-labeled PIP2 is specifically detected in freeze-fractured plasma membranes of both soma and dendritic areas of neurons.
a Representative TEM images of anti-PIP2 immunogold-labeled freeze-fracture replica of liposomes composed of 65% (w/v) PE, 30% (w/v) PC and 5% (w/v) of either PI(4,5)P2 (PIP2) (a), PS (b), PI(3,4,5)P3 (PIP3) (c), or PI(3,4)P2 (d). Arrowheads highlight examples of gold labels. Bars, 200 nm. e Quantitative analyses of labeling densities. PI(4,5)P2, n = 25; PS, n = 35; PI(3,4,5)P3, n = 21; PI(3,4)P2, n = 21 (for bar/dot plot presentation of the quantitative data in e, see Supplementary Figure 1). f–o TEM images (f, f’, f”, h–j, l–n) and quantitative analyses (g, k, o) of anti-PIP2 immunogold labelings of freeze-fractured plasma membranes of soma (f, f’, f”, g) and dendrites (h–n) of hippocampal neurons (DIV14-16). Control surface evaluations (E-face, ice) (g–k) and control experiments with antibodies quenched for specific binding by preincubation with liposomes containing PIP2 (i, k) demonstrated the specificity of labeling. (l–o) Additional quantitative experiments with different dilutions of anti-PIP2 antibodies establishing an antibody dilution of 1:100 as yielding saturated PIP2 detection. Bars, 200 nm. Soma P-face, n = 20; soma E-face, n = 12; ice, n = 14 ROIs. Dendrites: P-face, n = 40; E-face, n = 37; ice, n = 41; quench (P-face), n = 41 ROIs. Dendrites (P-faces) labeled with different antibody dilutions: 1:200, n = 78; 1:100, n = 78; 1:50, n = 82 ROIs. Data, mean ± SEM of at least two independent assays each. Statistical significance calculations, One-way ANOVA/Tukey´s Multi Comparison test (g), Kruskal–Wallis/Dunn’s (e, k, o). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. For P < 0.0001, exact P-values are not available. Other P-values are reported directly in the figure. For numerical source data, see Supplementary Data 1.
