Evc and Evc2 localize to the ciliary membrane in co-transfected IMCD3 cells. Each row consists of images from one representative cell. (a). Single transfection and co-transfection of Evc and Evc2-GFP constructs (the protein expressed is given on the left of each image). Expression of either protein alone does not result in localization on cilium. Evc is identified by S43B immunostaining (Evc ab, red), Evc2 by the GFP tag and cilia by acetylated tubulin staining (tubulin; green or red). Co-expression of both proteins results in their expression on the cilium. (cilia from two independent cells are shown). Merged images also show DAPI staining (blue). (b). Immunodetection of Evc and Evc2-GFP co-expressed in cells treated with (+) or without (-) Triton X100 to permeabilize the membranes and allow access to the antibodies. Evc ab (S43B), raised to Evc aa 459 - 999, does not detect Evc in non-permeabilized cells; Evc2 ab 1 (Y20), raised to a Evc2 peptide between aa 50 - 100, detects Evc2 GFP in non-permeabilized cells and Evc2 ab 2 (R1656), raised to Evc2 aa 780 - 1124, does not detect Evc2 in non-permeabilized cells. (c). Schematic representation of Evc and Evc2 on the ciliary membrane as determined from B, the C-termini of both proteins are intracellular. The regions for which we confirmed interaction are indicated by boxes and the predicted coiled-coil regions are shown as small black squares. Regions that are homologous (EVC: 339 - 889, and EVC2: 469 - 1018) are indicated by shaded rectangles. The epitope regions detected by Evc and Evc2 antibodies are indicated. Scale bar throughout 5 μm.