Ab69507

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).

The following antibodies were used in this study: RNF31/HOIP (ab46322, Abcam; 1:1000 western blot), GST (91G1, Cell Signaling Technology; 1:1000 western blot), GFP (A11122, Molecular Probes; 1:1000 western blot), RFP (Invitrogen; 1:3000 western blot), SHARPIN (ab69507, Abcam; 1:300 western blot), Anti-hamster α5β1 PB1 antibody (Developmental Studies Hybridoma Bank; 1:100 FACS), β-tubulin (ab6160, Abcam; 1:1000 western blot).
The secondary antibodies used in this study were Alexa 488- or Alexa 564-conjugated IgGs (immunofluorescence; Invitrogen) and HRP-conjugated IgGs (Far-Western analysis, ELISA assay and immunoblotting; GE Healthcare).

ChIP assay was performed as our previous described [8 (link)]. MCF7 cells were treated with vehicle or 10 nM estradiol for 30 minutes before crosslinking. The antibodies were used as follows: SHARPIN (AB69507, abcam), ERα (HC20, santa cruz) and rabbit IgG (sc2027, santa cruz). The sequences for ChIP primers were shown in Supplementary Table.