Confocal microscopic images of JAM1 in IHGE cells.

(A) Schematic view of the JAM1 construct. The Myc-mCherry tag is indicated by the magenta box, and the HA-tag is indicated by the green box. The signal peptide is indicated by the blue box, and IG-LIKE and TMD domains are indicated by the gray boxes and orange box, respectively. (B) Confocal microscopic images of an IHGE cell expressing Myc-mCherry-JAM1, stained with anti-JAM1 (green) and anti-HA (cyan). mCherry is shown in magenta. Higher magnifications of the areas indicated by the white boxes in the left panels are shown at right. Scale bar, 5 μm. (C–F) IHGE cells were transiently transfected with plasmids encoding Myc-mCherry–tagged HA-inserted JAM1; in (C) and (D), the cells were also transfected with EGFP-SEC61β (green). Following 48 h of incubation, cells were fixed, stained with anti-HA (D, magenta; F, green), and also stained with Alexa Fluor 488–conjugated phalloidin (E, green) or Alexa Fluor 633–conjugated phalloidin (F, magenta). The cells were then analyzed by immunofluorescence microscopy. Higher magnifications of the areas indicated by white boxes in the upper panels are shown on the lower side. Scale bars, 5 μm. See also S9A–S9D Fig.