Identification of a Novel Alternatively Spliced Form of Inflammatory Regulator SWAP-70-Like Adapter of T Cells

Figure 4

Effect of SLAT2 expression on T cell adhesion and filopodia formation. (a) Jurkat-TAg cells (5 × 10⁶) were transfected with empty vector, pEF-SLAT or pEF-SLAT2 (8 μg each). The cells were either left unstimulated or stimulated with 10 μg/mL anti-CD3 Ab (OKT3) for 45 min and subsequently analyzed for adhesion to plate-bound Fc-ICAM-1. Data represent means of triplicates, and graphs show means ± SD (left). Expression of transfected proteins was detected with an anti-SLAT Ab or anti-Myc Ab (right). Statistical analysis was performed using Student’s t-test (; NS, not significant). (b) Jurkat-TAg cells (5 × 10⁶) were transfected with pSI-EGFP vector, pSI-EGFP-SLAT, or pSI-EGFP-SLAT2 (8 μg each). The cells were stimulated with 2 μg/mL anti-CD3 Ab (OKT3), immobilized on poly-L-lysine-coated coverslips, and fixed and stained with Alexa Fluor 546 Phalloidin and DAPI. Representative confocal images of stimulated cells imaged in each group are shown (upper). Scale bar, 10 μm. Expression of transfected proteins was detected with an anti-SLAT Ab or anti-GFP Ab (lower). (c) Quantitative analysis of the imaging data in panel (b). Cells were categorized into three groups, namely, long filopodia, short filopodia, or no filopodia. Long filopodia are defined as having a length ~5 times longer than short filopodia [43]. The percentage of cells in each class is shown. >100 cells were counted in each group. Data represent means of triplicates, and graphs show means ± SD. Statistical analysis was performed using Student’s t-test (). Scale bar, 10 μm. Data are representative of three independent experiments.