AC mutation alters DP phosphorylation by GSK3. (A) GSK3 interactions with R2834H and WT DP S-tag (DP CT) in HEK 293 cells were examined following S-tag pulldown analyses where RIPA lysates (input) and pull-down products were analyzed by immunoblotting. (A′) Densitometry quantification of GSK3 interactions with WT and R2834H DP S-tag (DP CT). *, P < 0.03. Error bars indicate SEM. (B) PLA of WT, R2834H, or S2849G DP-GFP cells incubated with primary antibody pairs of GFP-GSK3. Nuclei were stained with DAPI (blue). (B′) PLA fluorescence intensity divided by number of nuclei was performed for each cell line using primary antibody pairs of GFP-GSK3 and with controls GSK3-RIgG or GFP-MIgG. Data quantification was analyzed from three separate experiments (>500 nuclei per experiment). *, P < 0.05. Error bars indicate SEM. (C) Control or caGSK3-transfected and LiCl- or Ly-294,002 (GSK3 Act.)–treated DP-GFP cell lines, assessed by immunofluorescence. Bars, 10 µm. (D and D′) R2834H DP S-tag (DP CT) cells were treated with NaCl or LiCl or transfected with caGSK3, assessed by immunoblotting. Densitometry quantification of three independent experiments. *, P < 0.001. Error bars indicate SEM.