Fig. 2. p190 Rho-GAP is required to mediate plexin signalling in fibroblasts. (A) Immunoblots of total cell lysates of NIH-3T3 fibroblasts expressing PLXNB1 and engineered with siRNAs targeted to p190 transcript or to an unrelated sequence. The filter was probed with specific antibodies directed against p190, PLXNB1 and actin. The endogenous levels of p190 are greatly reduced in NIH-3T3 fibroblasts by expression of targeted siRNA, while plexin expression is unaffected. (B) NIH-3T3 fibroblasts expressing PLXNB1 (or PLXNB1/A1) and engineered with p190-targeted siRNAs (or controls) were grown on glass coverslips and subjected to treatment with 5 mM Sema4D for 15 minutes. Cell were then analysed by immunofluorescence with EC-6.9 antibodies directed against the extracellular domain of PLXNB1. Scale bar: 20 µm. The average fraction of collapsed cells was determined in each condition (Barberis et al., 2004) and it is shown on the right. Plexin-dependent collapse response is greatly impaired in the absence of p190. (C) The attachment of the same cells as above to fibronectin-coated wells was assayed, in the presence or absence of 5 nM Sema4D. After 30 and 120 minutes, adherent cells were fixed and stained with crystal violet. Cell adhesion was eventually quantified by eluting the dye and measuring the absorbance at 595 nm. (D) In a similar experiment as described in C, we scored the average fraction of fibroblasts spread on fibronectin, after 1 hour incubation with or without the indicated amounts of Sema4D (see Barberis et al., 2004).