Fig. 7. p190 is recruited to plexins, and its GAP activity is induced upon receptor activation. (A) p190-GFP and either PLXNB1 or PLXNA1 (VSV-tagged) specifically co-precipitate from lysates of co-transfected 293T cells, whereas a truncated form of PLXNB1 lacking the cytoplasmic domain (PlexinB1-IC) does not associate with p190. Immunoprecipitations were performed using either anti-tag antibodies (GFP for p190 and anti-VSV for plexins, respectively) or non-related serum (nrs). Western blots were probed with the indicated anti-tag or protein-specific antibodies. Note that PLXNB1 is larger than other plexins (approx. 300 kDa), while PlexinB1-IC and PLXNA1 are almost identical in size (approx. 220 kDa). (B) Co-immunopurification of endogenous p190 with PLXNB1 transfected in 293T cells is induced after 5 minutes stimulation with 5 nM Sema4D. The expression levels of p190 and PLXNB1 in cell lysates are shown at the bottom. (C) Functionally active p190 was pulled-down (by means of constitutive active RhoQ63L-GST coated beads) from lysates of 3T3 fibroblasts expressing PLXNB1 and treated with 5 nM Sema4D for the indicated times. Immunoblotting with specific antibodies was used to detect p190 in pull-downs and in total lysates (included as loading controls). As measured by band density quantification, the level of activated p190 transiently increases upon plexin activation.