Mutation at actin binding site loses functions of F-actin stabilization and T cell activation. (A) Schematic diagram of wild type (WT)–TG2 and TG2ΔABM. CH, calponin homology; ABM, actin-binding motif; CR, calponin repeat. CHO-KI cells transfected with WT-TG2_GFP or TG2ΔABM_GFP were stained for F-actin (TRITC-phalloidin), and colocalization between GFP and TRITC was calculated with the FLUOVIEW and represented as a percentage (bottom). Images are representative of >50 cells examined. Results represent means ± SD of triplicate experiments. *, P < 0.05 compared with cells expressing WT-TG2. EV, empty vector. (B and C) F-actin binding (B) and actin depolymerization (C) assays were performed as described in Fig. 4 A and Fig. 5 D. Data shown are representative of three independent experiments. M, molecular mass; S, supernatant; P, pellet. (D) J-GFP, J-TG2_GFP, and J-TG2ΔABM_GFP were incubated with SEE-loaded Raji B cells for 30 min. (left) The arrows indicate the accumulation of GFP signals at the IS. The ratio of contact/opposite fluorescence was calculated. The ratio of contact/opposite fluorescence in the white boxed area was calculated. Each dot represents 50 cells examined. Horizontal bars indicate the mean of fluorescence intensities. Data shown are representative of three independent experiments. T, J-GFP, J-TG2_GFP, and J-TG2ΔABM_GFP cells; B, Raji B cells. (E) IL2 mRNA levels were measured using real-time quantitative RT-PCR. (F) Conjugate formations were determined as described in Fig. 3. (G) IL-2 secretions in each condition were assessed by ELISA. Results are the means ± SD of triplicate experiments. *, P < 0.05 versus J-GFP; **, P < 0.05 versus J-TG2_GFP. T, TAGLN2; C, cofilin. Bars, 10 µm.