Integrin-Binding Protein Interaction Study

Flag antibody (SG4110–16, Shanghai Genomics), 6 × his antibody (SG4110–06, Shanghai Genomics), sharpin antibody (ab174545 and ab69507, Abcam), FAK antibody (#3283, CST) and Y-FAK antibodies (#3285, CST) were used for immunoblotting; PAC1 antibody (340535, BD Biosciences), 9EG7 antibody (553715, BD Biosciences) and 7E2 antibody (DSHB) were used for FACS analysis. Plasmid of GST-fibronectin type III repeats 9–11 (GST-Fn-III) was kindly provided by David Calderwood [18 (link)]. The cDNA of full length sharpin was kindly provide by Ivan Dikic [26 (link)], and subcloned into vectors of pET28a, pHis-1, pGST-1 and pGADT7 for different experiments. The CT of integrin α5β1 and integrin αIIbβ3 were subcloned into pGST-1 vector. Kindlin-1 was subcloned into pET31b, pGST-1 and pGBKT7 vectors. To express and purify proteins, the expression vectors were transformed into Rosetta DE3 strain and induced to express proteins with 0.4 mM of IPTG. GST-tagged or his-tagged proteins were purified by Glutathione Sepharpose (GE) and Ni-NTA agarose (Qiagen) respectively, according to the manufactures’ protocols. The purified GST-Fn-III protein was further labeled with biotin (EZ-Link™ NHS-Biotin, Thermo Fisher).