FIG 2.
The Shc PTB domain binds the FAK FERM domain. (A) Cells transduced with either Flag or Flag-PTB were immunoprecipitated with anti-Flag and immunoblotted with antiphosphotyrosine (pTyr) or anti-Flag. HC, Ig heavy chain, LC, Ig light chain. (B) Cell lysates were immunoprecipitated with protein G-Sepharose (PGS), control Ig, or anti-Shc and immunoblotted for FAK and Shc. The bottom panel shows the results obtained with the lysate after immunoprecipitation, showing substantial capture of Shc. The results are representative of those from 3 immunoprecipitation experiments. Shc (IP), Shc in the immunoprecipitate; Irrel, irrelevant antibody. (C) Bacterially expressed His fusions of full-length p66Shc, p66Shc from which the PTB domain was deleted, or the isolated PTB domain were used to pull down endogenous FAK from cell lysates. The relative expression of His-Shc baits is 1:2.1:0.7. The results are representative of those from 2 experiments. (D) (Top) The cartoons depict serial deletions of the HA-tagged FAK transfected into HUVECs. His-tagged p66Shc was used to pull down cell lysates. Input and pulldown fractions for FAK and His-p66 bait are shown. The relative expression of the HA-FAK proteins is 1:1.1:1.1:1.4:1.6. (E) Cells transfected with full-length (FL) or FERM domain-only FAK-HA fusions were pulled down by His-p66. Pretreatment of lysates with PTP-1B is marked. Input and pulldown fractions were blotted for HA and Src. (F) Lysates of untransfected cells were used for pulldown by His-tagged wild-type p66 or p66 R285Q. Input and pulldown fractions for endogenous FAK are shown. (G) Fibronectin-coated beads were added to untreated or FAK inhibitor 14-treated (1 μM for 30 min) cells, and tension was applied. Adhesion complex and lysate fractions were blotted for Shc and FAK. The results in panels E to G are representative of those from 2 experiments. The numbers to the left and right of the gels in panels A to G are molecular masses (in kilodaltons). (H) HUVECs were plated on fibronectin, and PLA was performed. Primary antibodies (Ab) for FAK and Shc, Shc only, or FAK only were omitted for 3 negative controls, as shown. Bars, 20 μm.