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. Author manuscript; available in PMC: 2021 Sep 17.
Published in final edited form as: Mol Psychiatry. 2021 Jan 4;26(6):1775–1789. doi: 10.1038/s41380-020-00991-1

Fig. 2. Ankyrin-G 190 and Homer1b/c interaction in nanodomains within dendrites and spines.

Fig. 2

a. Western blot of subcellular fractionation from 16 weeks old mouse cortex is probed with ankyrin-G, Homer1b/c, N+/K+ ATPase α1 (as a plasma membrane marker), PSD-95 (as a postsynaptic marker), and β-actin antibodies. Each lane was loaded with 10 μg of each sample. WL, whole cell lysate; S1, cytosol/membranes; S2, cytosol; P2, crude synaptosomes; PSD, PSD-enriched synaptosomal fraction. b. Co-immunoprecipitation experiments with anti-ankyrin-G or anti-Homer1b/c from the PSD-enriched synaptosomal fraction. IgG, control IgG; IP, immunoprecipitation. c. Binding of ankyrin-G and its truncation mutants to Homer1c. The top panel shows the immunoprecipitated full-length ankyrin-G or the truncated versions of ankyrin-G co-expressed with Homer1c. d. Effects of point mutations in the PPXXF Homer-binding motif of ankyrin-G (P1606L and F1609R) on the interaction with Homer1c. The HEK293T cell lysate was analyzed by immunoblotting with HA or Flag antibody. n = 4 per each group. F(2, 9) = 24.10; F(2, 9) = 6.17; *, p < 0.05 ***, p < 0.001; followed by one-way ANOVA followed by a Bonferroni test. Data are represented as mean ± SEM. e. Schematic representation for the in situ proximity ligation assay (PLA). f. Confocal images for detection of the interaction between HA-ankyrin-G wild-type (WT) or point mutants (red) and Flag-Homer1c (green) with PLA (magenta) in COS7 cells. Scale bar, 20 μm (top and bottom). Bar graph of the PLA signal with the mutants of HA-ankyrin-G and Flag-Homer1c. n = 13 per each group. F(2, 36) = 6.48; *, p < 0.05, **, p < 0.01; one-way ANOVA followed by a Bonferroni test. Data are represented as mean ± SEM. g. SIM image of a mCherry-expressing neuron to detect the interaction between ankyrin-G and Homer1b/c by PLA (cyan). Scale bar, 2 μm (top panel). Zoomed images were shown from the boxed area in the top panel. Scale bar, 0.5 μm (middle and botton panels). h. Bar graph showing the PLA puncta ratio in spines versus dendrites, n = 9 cells. i. SIM image of mCherry-expressing neurons immunostained for anti-ankyrin-G (red) and anti-Homer1b/c (green). Scale bar, 2 μm. Colocalization is shown in white by ‘colocalization highlighter’ in ImageJ. j. High-resolution image of boxed spine in (i). Bottom panels: ratiometric images and colocalization (white). Scale bar, 0.5 μm. k. Pie chart showing the highlighted puncta ratio of spines versus dendrites per spine area. l. Pie chart of expression patterns of ankyrin-G and Homer1b/c in spines. m. Spine head size from the chart (k) was analyzed with a bar graph. F(3, 250) = 5.77; *, p < 0.05, ***, p < 0.001; one-way ANOVA followed by a Bonferroni test. Data are represented as mean ± SEM. n-p. Correlation plot of the size of anti-ankyrin-G (n) or anti-Homer1b/c (o) nanodomains versus spine head area, and the size of anti-ankyrin-G versus anti-Homer1b/c nanodomains (p). n = 13 cells. Head area: 0.452 ± 0.017 μm2; anti-ankyrin-G nanodomain area: 0.032 ± 0.002 μm2; anti-Homer1b/c area: 0.056 ± 0.003 μm2.