TAGLN2 is associated with F-actin and localizes at the d-SMAC of IS in T cells. (A) CHO-K1 cells transfected with TG2_GFP or GFP vector were stained with TRITC-phalloidin. The boxed areas (yellow) are represented as zoomed images in the right and bottom micrographs. (middle) The colocalization of GFP and TRITC signals was estimated by the Pearson’s correlation coefficient (R). (right) R values of single cells are represented as single dots, and ≥50 cells were examined. Horizontal bars indicate the means. *, P < 0.05 versus GFP-transfected cells. (B) J-GFP and J-TG2_GFP cells were stimulated with anti-CD3/28. Samples were immunoprecipitated and blotted with antibodies against the indicated proteins. Data shown are representative of three independent experiments. IP, immunoprecipitation; IB, immunoblot; M, molecular mass. (C) Mouse CD3⁺ T cells on anti-CD3/28 coverslips were stained for TG2 (rabbit IgG, negative control) and actin. (D) J-GFP and J-TG2_GFP cells transfected with LifeAct (Life-A) on PLL-coated coverslips were imaged and fluorescence intensity profiles were analyzed. Images and intensity profiles are representative of >50 cells. (E) J-TG2_GFP cells were conjugated for 30 min with SEE-Raji B cells (ICAM-1 [IC1]). 3D and rotating views of selected zoomed images (90°) of the boxed area are shown. Also refer to Video 1. Arrows indicate accumulation of TG2 and ICAM1 at the synapse site. (F) J-TG2_GFP cells on anti-CD3/28 coverslips were stained for LFA-1, Arp3, myosin IIA, and F-actin. Bottom images are shown, and each z-stack image is shown in Fig. S2. Also refer to Videos 2–4. c, central SMAC; p, peripheral SMAC; d, d-SMAC. All images are representative of >50 cells examined from three independent experiments. DIC, differential interference contrast. Bars, 10 µm.