Fig. 4. Immunoelectron microscopic analyses of migfilin clusters at cell-cell junctions. Immunoelectron microscopic analysis of HaCat cells was performed as described in Materials and Methods. Cell adhesions connecting the two cells are shown in the orientation between the two open arrows. Note that the anti-ß-catenin antibody (10 nm gold) and the anti-migfilin antibody (5 nm gold) were colocalized at areas of cell-cell contacts (between arrowheads). (B,C) Single pre-embedding immunotransmission microscopic images of HaCat cells at low (B) and high (C) magnification. In B, migfilin (beads) was clustered and associated with the actin bundles at cell-cell junctions (arrows). N indicates the nuclei of two neighboring cells. In C, desmosomes are indicated by arrows, and migfilin clusters are shown by arrowheads. Note that migfilin clusters were associated with the actin bundles but not desmosomes. (D) HaCat cells were prepared for pre-embedding immunotransmission microscopy and double stained with anti-migfilin and anti-ß-catenin antibodies. Note that migfilin (10 nm gold particles) and ß-catenin (5 nm gold particles) were closely clustered with the actin bundles.