Persistent PKA activation promoted Na _V 1.5/EB1 interactions in myocytes. (A) Detecting GFP-Na_V1.5 and EB1-mRFP interaction in ventricular myocytes by in situ proximity ligation assay (PLA). Shown are PLA signals and immunofluorescence of GFP-Na_V1.5 and EB1-mRFP in myocytes transduced with Adv-GFP-Na_V1.5 and Adv-EB1-mRFP and cultured for 24 h (low expression) and 48 h (strong expression), the latter under the control conditions or with PKA activation for 15 h. These images are z-stack projections of maxima. (B) Persistent PKA activation increased PLA puncta density. The images of z-projection of maxima were segmented in a systemic manner across CON and PKA myocytes to define PLA puncta, followed by calculating the percentage of cellular area occupied by PLA puncta. To correct for the differences in GFP-Na_V1.5 and EB1-mRFP expression level, the PLA puncta density of each of the myocytes was divided by the product of normalized GFP-Na_V1.5 and EB1-mRFP pixel content of the same myocyte from the same myocyte. Numbers in parentheses are myocytes studied from two independent experiments, with P value from t test between the two groups. (C) Top: Cartoon depicting plane of view. Bottom: 3-D views of PLA puncta in a CON myocyte, as a merge of PLA puncta (white), GFP-Na_V1.5 (green), and EB1-mRFP (salmon) or PLA puncta within cell contour (salmon line). Scale bars in all image panels are 10 μm.