Integrin Immunoblotting and Immunoprecipitation

Cell lysates were resolved by SDS-PAGE under non-reducing conditions, blotted onto PVDF or nitrocellulose membranes (Bio-Rad, Hercules, California, USA), and probed with rabbit monoclonal antibodies against integrins αv, αvβ3, αvβ5, αvβ6, or αvβ8 ([27] (link), kindly provided by Merck KGaA, Darmstadt, Germany). The antigens were detected using a peroxidase-conjugated secondary antibody (Thermo Fisher Scientific, Waltham, Massachusetts, USA) and SuperSignal WestFemto ECL reagent (Thermo Fisher Scientific). Chemiluminescence signals were captured with a GEL LOGIC 2200 imaging system and analysed with the Kodak MI SE software. For the detection of the αvβ1 complex immunoprecipitation was performed with a β1 antibody (Millipore clone P4C10, Merck KGaA). The immune complexes were eluted in non-reducing sample buffer, resolved by SDS-PAGE, immunoblotted and detected with the rabbit monoclonal antibody against αv.