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Immunoblotting and quantification

IR Ignacio Rodriguez-Pastrana
EB Eleni Birli
AC Amanda S. Coutts
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Cells were seeded into 6 cm dishes and treated as appropriate before harvesting in TNN buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.4, 5 mM EDTA, 0.5% NP40, 50 mM NaF, 0.2 mM Na3VO4, in the presence of protease inhibitors). Protein quantification was performed using Bradford Reagent (Sigma-Aldrich) and lysates were run using SDS-PAGE, transferred to nitrocellulose membranes and probed overnight at 4 °C with primary antibodies diluted in 5% skimmed milk in 0.1% Tween-20 in PBS (v/v). Membranes were washed extensively and probed with secondary antibodies before detection by enhanced chemiluminescence (ECL) using ChemiDocTM XRS+ with Image LabTM software (Bio-Rad). Band quantification was performed using Fiji/ImageJ [36]. Uncropped blots are presented in Fig. S8.

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