Fig. 4. Y731 within the intracellular domain of VEC is important for lymphocyte migration. Mouse endothelioma cell lines, null for VEC and stably re-expressing wt or Y to F mutants of VEC were grown to confluence. (A) Equal amounts of proteins were analyzed by immunoblotting using anti-VEC and anti-ERK antibodies. The position of size markers (in kDa) is indicated on the left. (B) Immunocytochemical analysis of the VEC distribution. Bar, 10 µm. (C) Mouse endothelioma cell lines, null for VEC, stably re-expressing wt VEC or transiently nucleofected with the VEC-GFP-expressing plasmid pEGFP-N'-VEC were grown to confluence. They were then incubated with antigen-specific T cells, which were allowed to adhere and migrate for 4 hours. Adhesion (white) and migration (black) across these EC populations were then determined as described in the Materials and Methods section. Results are expressed as the percent increase of VEC-null EC (mean ± s.e.m. of six replicates from five independent experiments). (D) Lymphocyte migration across the indicated stable mouse endothelioma cell lines. Adhesion (white) and migration (black) across individual transfected EC populations were then measured as above. Results are expressed as % of control cells re-expressing wt VEC (mean ± s.e.m. of six replicates from at least three independent experiments). Significant differences were determined by Student's t-test (^*P<0.005, ^**P<0.0001).