Both plexin-A1 and plexin-A4 are required for sema3A-induced cell contraction. (A) HUVECs expressing sh-control or sh-plexins were incubated for 15 minutes with conditioned medium derived from HEK293 cells infected with lentivirus expression vector (control) or with lentiviruses directing expression of sema3A (sema3A), or sema3E (sema3E). Cells were fixed and stained for the intracellular distribution of vinculin (arrows). (B) U87MG cells expressing sh-plexA1 or sh-plexA4 (bottom) were incubated with control or sema3A containing conditioned medium for 15 minutes. Actin filaments were then visualized using fluorescent phalloidin (top). (C-D) PAE cells coexpressing myc tagged plexin-A1 and neuropilin-1¹⁵  were infected with a lentivirus directing expression of V5 tagged plexin-A4. The infected cells were lysed, (C) plexin-A4 was immunoprecipitated using anti-V5 antibody, and the lysates examined for the presence of plexin-A1 using anti-myc antibody. (D) Plexin-A1 was immunoprecipitated from cell lysates using anti-myc antibody and the presence of plexin-A4 in the lysates was examined using an anti-V5 antibody. Immunoprecipitation with an anti-HA antibody was performed as a negative control. Shown is a representative experiment of 3 that gave similar results. (E) ELISA assay using the extra-cellular HIS-tagged human plexin-A4 coated wells and increased concentrations of the extra-cellular FC-tagged mouse plexin-A1.