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Figure S5. p190RhoGEF can be phosphorylated by Pyk2, FAK, or Src in vitro. Recombinant GST-Pyk2 414–689, GST-FAK 411–686, and His-Src were generated using the Baculogold system (BD Biosciences), FPLC purified, concentrated, and stored frozen in kinase buffer (20 mM tris, pH 7.4, 400 mM NaCl, 0.5 mM Na₃VO₄, 25 mM MgCl₂, 5 mM MnCl₂, 1 mM EDTA, 5 mM β-mercaptoethanol, and 5% glycerol). Purity was estimated at >90% by SDS-PAGE and Coomassie blue staining. GFP-190RhoGEF was transiently expressed in 293T cells, and anti-GFP polyclonal IPs from either mock- or GFP-p190RhoGEF–transfected cells were washed three times and incubated in kinase buffer in the presence or absence of 100 ng of recombinant Pyk2, FAK, or Src kinases. In vitro kinase assays were initiated by the addition of 20 µM ATP with 20 µCi/nmol [³²P]ATP, incubated at 32°C for 15 min, and resolved by SDS-PAGE. Protein phosphorylation was visualized by autoradiography.