Roles of ROCKs and GEF-H1 in T cell polarity on EC. (a) p-MLC levels in CEM cells. GAPDH, protein loading. Representative of four experiments. (b and c) Polarity quantification after 30 min on EC for siRNA-transfected (48 h) or Y27632-treated (10-min preincubation and continued presence) CEM cells (b, n = 3; c, n = 5; 100 cells/experiment). (d) Localization of F-actin, α-tubulin, and ICAM-3 after 30 min on EC in Y27632-treated cells (10-min preincubation and continued presence). Arrows, long tails; arrowheads, lateral protrusions. (e) Process of CEM cell protrusion formation on EC determined from 1-h time-lapse videos (n = 40 cells/condition; three experiments). (f) GEF-H1 localization in CEM cells after 30 min on EC. LE, leading edge; U, uropod. (g) CEM cell length after 30 min on EC (n = 3; 50 cells/condition/experiment). *, P < 0.05; **, P < 0.01. Error bars indicate mean ± SEM. Bars, 10 µm.