Figure 3.

Evidence for interaction of endogenous PP2A and GABA_BR in cultured cortical neurons. A–C, Representative PLA images of cultured mouse cortical neurons (DIV 6) with the indicated primary antibodies. Top three panels (Ai, Bi, Ci) show PLA signal (red) between PP2A-C subunit and GABA_BR1, PP2A-B55 and GABA_BR1, and between GABA_BR1 and GABA_BR2 subunits. Bottom three panels (Aii, Bii, Cii) show corresponding negative controls where a primary antibody was omitted. Green is MAP2 immunostaining; red is PLA signal; blue is DAPI staining. D, Scatter plot showing quantification of PLA signal corresponding to A–C from one representative experiment. Each data point represents analysis of one image containing ∼80 neurons. Magenta bar indicates mean. ****p < 0.0001 using unpaired Student's two-tailed t test. E, F, Regulation of PP2A-C and GABA_BR1 interaction by intracellular Ca²⁺. E, Representative images of DIV 6 cortical neurons treated with vehicle (Ei) or the calcium ionophore A23187 (2 μm, Eii) for 30 min before PLA for PP2A-C and GABA_BR1. F, Scatter plot showing normalized PLA signal pooled from three independent experiments. Data points were normalized to the vehicle average for each experiment. Control group received vehicle treatment but the PP2A-C antibody was omitted. Red bar indicates group mean for three experiments (****p < 0.0001, one-way ANOVA with Tukey's post hoc test; F_(2,81) = 167.8).