Figure 3

Localization of CALHM1/CALHM3 in the basolateral membrane of MDCKII epithelia. (A–E) Fluorescence double-labeling of GFP (green) and Na⁺/K⁺ ATPase (red) in polarized MDCKII cells that were transiently transfected with CALHM1-GFP and untagged CALHM3 and treated with 100 µg/mL cycloheximide (CHX) for the indicated time. (F) Double-staining of GFP (green) and Na⁺/K⁺ ATPase (red) in MDCKII epithelia transfected with untagged CALHM1 and CALHM3-GFP and treated with CHX for 2 h. Na⁺/K⁺ ATPase signals were used as a marker for the basolateral membranes. Upper panel, XY image. Lower panel, XZ image reconstructed at the blue line in the upper image. Note that Transwell filter membranes exhibit autofluorescence in the green channel and generate straight signals at the bottom of the cells in the XZ images throughout the manuscript. Api, apical membrane; Baso, basolateral membrane; Scale bars, 10 µm. Data are representative of two experiments.