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. 2009 Oct 5;29(23):6321–6334. doi: 10.1128/MCB.00103-09

FIG. 2.

FIG. 2.

Effects of the plexin-B1(Y1708F/Y1732F) mutant on plexin-B1 signaling. HEK 293 cells were transfected with cDNAs encoding wild-type (WT) VSV-tagged plexin-B1 [(VSV)-plxB1] or its mutant (Y1708/1732F) together with either Myc-tagged RhoA [(Myc)-RhoA] and wild-type PDZ-RhoGEF (PRG) (A), R-Ras, Rnd1, and PRG (B), or the constitutively active GST-tagged Rac1 mutant [Rac(Q61L)] and wild-type PRG (C). After 48 h cells were kept in 0.5% FBS (A) or 10% FBS (B and C) for 12 h followed by incubation without (−) or with (+) 150 nM Sema4D for 20 min. Thereafter, cells were lysed, and plexin-B1 was immunoprecipitated (IP) using anti-VSV-Sepharose (A and C). (A) Precipitated proteins were analyzed by immunoblotting using antiphosphotyrosine antibodies. In a parallel experiment active RhoA was precipitated from cell lysates as described in Materials and Methods. (B) Activated R-Ras was precipitated from the cell lysates with a GST-fused Ras-binding domain of Raf1. Shown are autoluminograms of immunoblots (IB) of lysed (lysate) or precipitated samples (IP/GST-RBD or GST-Raf1 pulldown) and the indicated antibodies.