Horseradish peroxidase hrp coupled secondary antibody

Protein lysates and western blots were performed as described [6 (link)] using FLAG (Sigma), ILEI [11 (link)], Erk1, beta-actin and vinculin (Cell Signaling Technologies) primary antibodies and horseradish peroxidase (HRP)-coupled secondary antibodies (Jackson ImmunoResearch).

Immunoprecipitation-RIPA or RIPA lysis buffer containing 1% phosphatase inhibitor cocktail A and B, protease inhibitor cocktail (Biovision, Milpitas, CA, USA), and 1% PMSF (10 mM) were used to extract proteins from cells and tissues, respectively. To collect the total protein, the extractions were centrifuged at 12,000× g at 4 °C for 5 min. The protein concentration was estimated using a BCA kit (Thermo Fisher Scientific, Waltham, MA, USA), and the proteins were heated at 98 °C with LDS sample loading buffer and 5% β-mercaptoethanol. Equal amounts of protein samples were resolved by 10% SDS polyacrylamide gel electrophoresis and transferred onto an immunoblot nitrocellulose membrane (Millipore, Burlington, MA, USA). After blocking in 5% skim milk for 1–2 h at room temperature, the membranes were incubated with specific primary antibodies and horseradish peroxidase (HRP)-coupled secondary antibodies (Jackson ImmunoResearch Inc., West Grove, PA, USA), respectively. The blot findings were visualized with ECL detection reagent and a Bio-Rad Imager (Bio-Rad, Hercules, CA, USA). The primary antibodies used in this study are listed in Table S2.

Protein extraction was performed using minimal amounts of RIPA buffer (Thermo Fisher Scientific, Cat No. 89901) containing 1x protease inhibitor (Merck, cOmplete^® protease inhibitor cocktail) and on ice incubation for 15min followed by brief sonication and a centrifugation step (13.000 ×g, 5 min, 4 °C) to remove cellular debris. iProtein extracts were resolved in NuPage^TM 4–12% BisTris Protein Gels (Cat No. NP0321BOX, Thermo Fisher Scientific, MA, US), and blotted onto an Invitrolon^TM PVDF (Thermo Fisher Scientific, Cat No. LC2005) or a Nitrocellulose membrane (Lifetech, Cat No. IB23001) according to standard protocols. After incubation with 5% nonfat milk in TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween 20) for 60 min, the membrane was rinsed with TBST and incubated with primary antibodies (Supplementary Table 2, online resource). Membranes were washed three times for 10 min and incubated with horseradish peroxidase (HRP)-coupled secondary antibodies (Jackson ImmunoResearch) for 1h at RT. Blots were washed with TBST three times, once with TBS, developed with a chemiluminescent substrate (ThermoFisher) and imaged with the ImageQuant 350 scanning system (cooled-CCD camera, GE Healthcare).