Figure 2

Role of the cytoskeleton in flotillin- 2 and PSGL-1 capping in human T-lymphoblasts in suspension. (A,B) After an overnight incubation in the absence of IL-2, T-lymphoblasts were incubated for 45 minutes at 37°C (control) or were preincubated for 30 minutes without or with latrunculin A (lat; 1 μM) or jasplakinolide (jasp; 5 μM) or blebbistatin (ble; 100 μM) or Y-27632 (Y; 10 μM) respectively followed by stimulation with 40 ng/ml of SDF-1 for 15 minutes at 37°C. Cells were then fixed with 10% TCA and (A) double-labeled either for flotillin-2 (flo2; rabbit Ab) and PSGL-1 (murine Ab) or (B) for flotillin-2 (flo2, rabbit Ab) and actin (murine Ab). Bar, 10 μm. (C-E) T-lymphoblasts were treated as described in (A) followed by evaluation of the % of polarized cells with contracted uropods (C); or evaluation of the % of cells with flotillin-2 caps (D) or evaluation of the % of cells with PSGL-1 caps (E). Mean ± s.e.m. of 3-7 independent experiments. At least 40 cells per sample and experiment were analyzed.