MEFs prespread on a recombinant integrin ligand (50K) were stimulated with heparin-binding fragments of fibronectin (H/0) before preparing lysates and then measuring levels of soluble p190-A in TrX lysates by Western blotting

Wild-type (A) and syndecan-4–null (B) MEFs reexpressing a Y188L mutant syndecan-4 defective in PKCα binding to endogenous levels, treated with 200 nM of the pharmacological PKC inhibitor BIM-1 (C), transfected with a nontargeting siRNA (D), and transfected with an siRNA targeted against PKCα (E). (F) Expression of PKCα after transfection with control or targeting siRNA. (G) Phosphotyrosine blots of p190-A immunoprecipitated from high-detergent lysates of MEFs expressing wild-type or Y188L-syndecan-4 spread on 50K and stimulated with H/0 for 10 min. (H) Phosphorimager scan and blot of p190-A immunoprecipitated from high-detergent lysates of MEFs expressing wild-type or Y188L–syndecan-4, metabolically labeled with [P]orthophosphate spread on 50K, and stimulated with H/0 for 10 min. RhoA activity ELISAs of MEFs expressing wild-type (I) or Y188L–syndecan-4 (J) prespread on 50K and stimulated with H/0. Equal loading between time points was confirmed by blotting for HSP70 or a total protein assay. Error bars represent standard error. *, significant change (P < 0.05) from experiments repeated on at least five separate occasions.<p><b>Copyright information:</b></p><p>Taken from "p190RhoGAP is the convergence point of adhesion signals from αβ integrin and syndecan-4"</p><p></p><p>The Journal of Cell Biology 2008;181(6):1013-1026.</p><p>Published online 16 Jun 2008</p><p>PMCID:PMC2426943.</p><p>