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Fig. 5. GFP-ß2-chimaerin-expressing Jurkat T cells show reduced CXCL12/PMA-induced Rac activation and adhesion to immobilized VCAM-1. (A) Control Jurkat cells (JK) or Jurkat cells stably transfected with GFP-ß2-chimaerin (JKß2) were plated onto fibronectin and stimulated with CXCL12 (200 ng/ml, 60 minutes) or PMA (100 ng/ml, 30 minutes). Cells were lysed and Rac-GTP determined by pull-down assays with PAK-RBD-GST (see Materials and Methods). Samples were separated by SDS-PAGE and Rac-GTP levels determined by western blotting with anti-Rac antibody. One-tenth of each sample was analyzed for total Rac level and ß2-chimaerin expression. (B) Jurkat cells were transfected with GFP or GFP-ß2-chimaerin-expressing vectors; GFP-positive cells were sorted after 24 hours. Sorted cells were added to wells coated with BSA or sVCAM-1, either alone (medium) or co-immobilized with CXCL12. Where indicated, cells were stimulated with PMA (100 ng/ml, 5 minutes) before addition to sVCAM-1-coated wells. Cells were allowed to adhere at 37°C for 2 minutes. Unbound cells were washed and the extent of adhesion determined as described (see Materials and Methods). Data represent the mean ± s.d. of at least three independent experiments performed in triplicate.
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