GSK3 phosphorylation of DP altered in R2834H DP mice. (A) Transgenic mouse hearts were lysed in urea sample buffer and phosphorylation of WT and R2834H DP-Flag were analyzed by immunoblotting using DP phospho-specific and GSK3 substrate phospho-specific antibodies. Total levels of DP expression were assessed using anti-DP (NW6) and Flag. Tubulin was used as a loading control. (A′) Densitometry quantification indicates that although total levels of DP expression are similar, phosphorylation of R2834H DP was dramatically decreased compared with WT DP using both the DP-specific phospho-antibody and a GSK3 substrate-specific phospho-antibody. The data shown are from a single representative experiment out of three repeats (n = 3). Error bars represent SEM. *, P < 0.001. (B) R2834H DP-trangenic mice demonstrate altered cytoplasmic localization of DP and disruption of cell–cell contacts in cardiac tissues, assessed by confocal immunofluorescence. WT and R2834H DP cardiac sections were stained with anti-Flag and anti-desmin antibodies. (C) PLA analyses were performed with WT and R2834H DP cardiac sections incubated with primary antibody pairs DP (NW6)-GSK3, DP (NW6)-MIgG, or GSK3-RIgG. Colocalization allows for hybridization, ligation, and amplification of oligonucleotide adducts fused to secondary antibodies, ultimately producing a fluorescent spot (red) in situ. Blue DAPI staining marks nuclei. Bars, 10 µm. (C′) For the quantification of PLA, PLA spots were counted and divided by total number of nuclei in a frame. Quantification shows a statistically significant enhancement of signal for the DP-GSK3 antibody pairing in WT hearts compared with R2834H transgenic mouse hearts. *, P < 0.03; **, P < 0.001; from >500 cells counted from four independent experiments. Error bars indicate SEM.