Plexin-mediated Sema3A stimulation induces cytoskeleton reorganization and redistribution of actin-linking proteins into lipid rafts. (A) Jurkat cells were incubated with control IgG or Sema3A-Fc (150 ng/mL) for 30 minutes. As positive control, Jurkat cells were also incubated with 50 ng/mL rhFasL. Actin morphology was visualized by staining with phalloidin-TRITC. Nuclei were stained with Hoechst 33 258 (AX10 microscope, Carl Zeiss, Oberkochen, Germany, original magnification, ×1600). (B) Jurkat cells were pretreated with 5 μg/mL cytochalasin B (CB) for 30 minutes and then incubated with anti-Fas or anti-Fas plus Sema3A-Fc for 6 hours. Cell death was quantified by flow cytometry with annexin V staining. P values were also shown. N.S., not significant. (C-E) Control or dominant negative plexin-A1 expressing cells (DN-plexin-A1) were treated with control IgG or Sema3A-Fc for 60 minutes and subjected to density gradient fractionation. (C,D) Fractions were immunoblotted with antibodies for G_M1, Ezrin, RhoA, and RhoGDI. Western blots were also probed with anti-VSV-G antibody to detect the VSV-G epitope tag of plexin construct. (E) Fractions derived from DN-plexin-A1 were immunoblotted with antibodies for Lck and Fas. Lipid raft (R) and soluble (S) fractions are indicated.