Pkp4- and p120-enriched AJs associate with different sets of proteins. (a and b) Maximum-intensity projections of all x–y optical slices of A431 cells double-stained for p120 or pkp4 (green) in combination with apical/basal AJ markers, vinculin, and afadin (a) or lateral AJ markers, erbin, and PLEKHA5 (b). Only merged images are shown for low magnifications. Bar, 20 μm. The zoomed regions indicated by white dashed boxes are shown in both channels at the bottom. Bar, 12 μm. (c and d) Corresponding scatterplots of red and green relative pixel intensities for the images in a and b. The Pearson’s r values are shown at the upper right of each plot. Note that substantial populations of p120/vinculin and p120/afadin pixels exhibit strong positive correlation indicated by shaded area between the red lines. Such pools of positively correlated pixels are undetectable for pkp4/vinculin or pkp4/afadin combinations. Instead, these combinations display two independent pixel populations distributed along the x and y axes. By contrast, pkp4 shows a stronger relationship with the lateral AJs markers, PLEKHA5 and erbin. (e) Average Pearson’s colocalization values (r) of images stained as in a and b. Four independent images were quantified for each marker pair. The means ± SD are indicated by bars. Note that apical AJ markers (vinculin and afadin) show substantially higher r values with p120 than with pkp4, whereas the opposite trend is observed for the lateral AJ markers (PLEKHA5, erbin).