a. SIM images to detect the interaction between HA-ankyrin-G WT or mutants (P1606L or F1609R) and anti-Homer1b/c by in situ PLA. The PLA was conducted after transfection of HA-ankyrin-G WT or mutant constructs into the cultured mouse cortical neurons. Negative control was transfected with only mCherry construct. Scale bar, 2 μm. b. The number of PLA puncta per 10 μm from HA-ankryn-G WT, P1606L, and F1609R transfected neurons. PLA was performed with anti-Homer1b/c and anti-HA antibodies. F(2, 123) = 11.75. c. Spine head size from mushroom spines was analyzed from only mCherry or HA-ankryn-G WT, P1606L, and F1609R transfected neurons with mCherry. F(3, 1445) = 18.62. d. Total numbers of all spines were counted. F(3, 141) = 3.64. e. Mushroom, thin, and stubby types of spine morphology were analyzed. F(6, 423) = 5.59. n = 13 (only mCherry), 17 (WT), 19 (P1606L), 21 (F1609R) cells. *, p < 0.05; **, p < 0.01; ***, p < 0.001; one-way ANOVA followed by a Bonferroni test for (b-d) and two-way ANOVA followed by a Bonferroni test for (e). Data are represented as mean ± SEM.