IGSF4 localizes at the c-SMAC in the immunological synapse. (A) J-IGSF4_GFP cells were incubated with Raji B cells stained with orange CMRA in the presence or absence of SEE. Arrowheads indicate the polarized IGSF4 (green) at the cell–cell contact sites. The percentage of T–T or T–B conjugates with surface IGSF4 relocation at the contact zone relative to the total number of conjugates in the absence or presence of SEE was analyzed from a total of 150 conjugates of each category. The results are the mean ± SD of triplicate experiments. The boxed areas (blue and orange) are represented as zoomed images in the right panels. (B) J-IGSF4_GFP cells were stained with anti-CD3 (cy5), anti-CD45 (cy5), or anti–LFA-1 (cy5) Fabs and then incubated with SEE-loaded Raji B cells stained with anti–ICAM-1 (cy3) Fab. See also Video 1. The data are representative of four independent experiments. The panels on the right represent zoomed images of the boxed areas (yellow) indicated in the differential interference contrast images. Bars, 10 µm.