ITGB7 binding to FN activates FAK, Src, and Rac-1. (A) Lysates from ITGB7^silenced and ITGB7^positive MM1S and H929 cells cultured on FN were screened by immunoblotting for the activation of FAK (phospho-Tyr397-FAK), Src (phospho-Tyr416-Src), and ERK (phospho-Thr202/Tyr204ERK1/2) and loading controls. (B-C) p-FAK localization and distribution in ITGB7^positive (B) and ITGB7^silenced (C) H929 cell cultures on FN-coated plates and measured by laser scan confocal microscopy. Shown are the images of immunostaining with anti-actin (Red: AlexaFluor 555 phalloidin), anti-phospho-FAK (Green: AlexaFluor 488) and DAPI with overlay images. (D) Rac-1 activation (GTP-Rac1 bound to GST-PAK1-p21-binding domain corresponding to residues 67-150) measured by immunoblotting with Rac-1 Ab on GST-PAK1 immunoprecipitated protein lysates (500 μg) from ITGB7^silenced and ITGB7^positive MM1S cells cultured under the indicated conditions (RP = regular uncoated plate, FN = fibronectin-coated plates) with anti-GST (Cell Signaling Technology) loading control. (E) Activation of Rac1 was measured in lysates from ITGB7^silenced and ITGB7^positive MM1S and H929 cells cultured on FN. Pulldowns of activated Rac1 (GTP-Rac1 bound to GST-PAK1 beads) were analyzed by Western blot for Rac1 and reprobed with anti-GST as loading control. Blot on the left represent a Western blot for total Rac1 in 10% of the imput lysate used for the immunoprecipiation of activated Rac1. Rac1-nS1 indicates the up-shifted band of Rac1 corresponding to SUMOylated Rac1. (F) Pulldowns of activated Rac1 (GTP-Rac1 bound to GST-PAK1 beads) from H929 ITGB7^silenced and ITGB7^positive cells cultured on FN were blotted with anti-SUMO1 Ab. Blot on the left represent a Western blot for total Rac1 in 10% of the input lysate used for IP. GTP-Rac1-nS1 indicates the up-shifted band of Rac1 corresponding to SUMOylated Rac1.