Persistent PKA activation for 15 h increased the size and density of Na _V 1.5, EB1, and β-Tub clusters at ICDs. (A) Procedures of analyzing clusters at ICDs. Top: Orthogonal views of cell ends of CON and PKA myocytes, showing merged immunofluorescence signals of nCadherin (nCad, blue), Na_V1.5 (green), EB1 (white), and β-tub (red). Middle: YZ-plane views of individual immunofluorescence, with ROI demarcated by nCad (white dotted lines). Bottom: Signals outside ROIs were cleared (black background), and signals inside ROIs were segmented to define clusters (black clusters on white background). This was followed by quantification of cluster density (% ICD area occupied by clusters), average size (µm²), and mean immunofluorescence intensity in clusters. (B) Box plots of cluster parameters for Na_V1.5, EB1, and β-Tub at ICDs of CON and PKA myocytes. Data are pooled from four independent experiments. For each myocyte, 10–12 YZ-plane images at 0.25–0.5 µm intervals, advancing from the cell end toward the cell center (before nCad signals disappeared), were exported to ImageJ for analysis. Numbers shown in B are those of ROIs quantified. Listed P values are from t tests between CON and PKA myocytes. In A, scale bars are 2 μm for the top row and 1 μm for the middle and bottom rows.