Establishment of Jurkat cell line stably expressing DOCK2 and DOCK2-dCS.

(A) Expression levels of DOCK2 of proteins were examined by immunoblotting with anti-Flag mAb. Lane 1, parental Jurkat cells; lane 2, Jurkat cells with vector control; lane 3, Jurkat cells with DOCK2-WT; lane 4, Jurkat cells with DOCK2-dCS. (B) Measurement of active form of Rac1 using established cell lines. For the positive control, Jurkat cells were preincubated with phorbol 12-myristate 13-acetate (PMA; Sigma) (1 μM) for 10 minutes. Cells were lysed and incubated with GST-PAK2-RBD and glutathione-Sepharose beads. The bound proteins were separated by SDS-PAGE, and endogenous Rac1 was detected by immunoblotting with anti-Rac1 mAb (B&D Transduction Laboratories). The intensities of precipitated Rac1 were measured and are depicted as a bar graph comparing each with the value of nontransfected control as 1.0. (C) Assessment of cell adhesion. Cells were plated on collage type I–coated (top panels) or poly-L-lysine–coated (bottom panels) dishes, and incubated at 37°C for 20 minutes. Cells were fixed with 4% paraformaldehyde for 10 minutes at room temperature and observed by phase contrast microscopy. Original magnification × 400.