Involvement of Plexin in Sema3A signaling. (A) Jurkat cells were treated with control IgG or Sema3A-Fc for 60 minutes and subjected to density gradient fractionation. For anti-Lck immunoblots, 10 μg of protein was loaded per lane; and for antiplexin-A1 immunoblots, 40 μg of protein was loaded per lane. (B) Jurkat cells (10⁷ cells per test) were treated with control IgG or Sema3A-Fc (150 ng/mL) for 5 minutes and lysates were prepared. Equivalent amounts of whole-cell lysates were either immunoprecipitated with antiplexin-A1 antibody (IP, antiplexin-A1) or anti-NP1 antibody (IP, anti-NP1). Immune complexes were then immunoblotted (Blot Ab) as indicated. Data in panels A and B are representative of 3 experiments. (C) The Jurkat cell line was engineered by lentiviral-mediated gene-transfer of a truncated form of plexin-A1, lacking its cytoplasmic domain (DN-plexin-A1). These cells or control Jurkat cells were incubated with CH11 or CH11 plus Sema3A-Fc for 6 hours. (D) In addition, Jurkat cells transfected with siRNA specific for plexin-A1 or nonsilencing siRNA were cultured with control IgG or Sema3A-Fc for 6 to 8 hours. Plexin-A1 expression in RNAi-treated Jurkat cells was determined by RT-PCR analysis. Cell death was quantified by flow cytometry with annexin V staining. P values were also shown. N.S., not significant.