TAGLN2 antagonizes cofilin in vivo, and TAGLN2^−/− T cells show substantial retardation of cofilin activity upon TCR stimulation. (A) CHO-K1 cells were transfected with the indicated constructs and then stained with Alexa Fluor 647–phalloidin. The integrated fluorescence (Fl) of F-actin in each transfected cell was estimated by FLUOVIEW. (B) Jurkat T cells were cotransfected with the indicated constructs. Cells were loaded on anti-CD3/28–coated coverslips for 5 min, fixed, and stained with Alexa Fluor 647–phalloidin. Images are representative of >50 cells examined. The areas and fluorescence intensity of F-actin were analyzed by FLUOVIEW. Data are means ± SD of triplicate experiments. (A and B) *, P < 0.05 versus empty vector (EV). **, P < 0.05 versus cofilin alone. (C) Live images of TG2_GFP and cofilin/S3A_RFP were acquired from cells in B. The colocalization (yellow and blue circles) was estimated as described in Fig. 1 A. The yellow arrows indicate the F-actin hole. The boxed area (yellow) is represented as 4× zoomed images in the right and quantitated by fluorescence intensity. Data are representative of >50 cells examined. (D–F) Indicated CD3⁺ T cells were stimulated with an anti-CD3/28 for the indicated time points. (D) The phosphorylated and total forms of cofilin were assessed by Western blotting. The phosphorylated and total forms of PI3K and ERK (E), Lck, Zap70, PLC-γ, and PKC-θ (F) were also detected by Western blotting. The data are representative of three independent experiments. DIC, differential interference contrast; M, molecular mass. Bars, 10 µm.