Figure 3.
Roles of myosin II and Rho-kinase in flotillin capping in human T-lymphoblasts adhering to fibronectin. A: After an overnight incubation in the absence of IL-2, T-lymphoblasts (1.5 × 106cells per well) were placed on fibronectin-coated glass coverslips without or with blebbistatin (ble; 100 μM) or Y-27632 (Y; 10 μM) respectively and allowed to adhere for 30 minutes at 37°C. To part of the samples, 40 ng/ml of SDF-1 was added followed by a further 30 minutes incubation at 37°C (total incubation 60 minutes). Cells were then fixed with 10% TCA and double-labeled for flotillin-2 (flo2; rabbit Ab) and flo1 (murine Ab). Bar, 10 μm. Asterisks indicate cells shown enlarged in the panels labelled "detail". B, C: T-lymphoblasts were treated as described in (A) followed by evaluation of the % of polarized cells with contracted uropods (B); or evaluation of the % of cells with flotillin-2 caps (C). Mean ± s.e.m. of 3-4 independent experiments. At least 40 cells per sample and experiment were analyzed. *P < 0.025, **P < 0.0125, ***P < 0.0005 for differences between data obtained for cells incubated in the absence and presence of inhibitors.