Transblot turbo pvdf

For the preparation of total protein extracts, cells were scraped, harvested and lysed in RIPA buffer (50 mM Tris-HCl, pH 8, 150 mM NaCl, 1% Triton X100, 0.5% DOCA, 0.1% sodium dodecyl sulfate (SDS)) supplemented with protease inhibitors (104 mM AEBSF, 1.5 mM pepstatin A, 1.4 mM E-64, 4 mM bestatin, 2 mM leupeptin, and 80 µM aprotinin) for 30 min on ice, then sonicated for 15 s, and centrifuged at 10,000 × g for 10 min at 4 °C. Proteins were quantified using the Bradford method and then proteins (25–40 μg) were separated on TGX acrylamide gels (1610172, Biorad) at 300 V for 30 min using and transferred onto Transblot turbo PVDF (1704157, Bio-Rad) membranes for 10–15 min with the Transblot turbo (1704150, Biorad) according to the manufacturer’s recommendations. Membranes were saturated in TBS-Tween 20 0.1% supplemented with 5% milk for 1 h and then incubated with primary antibodies (listed in Supp. Table 9) overnight at 4 °C. Membranes were washed 3 times with TBS-Tween 20 0.1% and incubated with secondary anti-rabbit or anti-mouse horseradish peroxidase conjugate antibody according to the manufacturer’s instructions (BI2407, BI2413C, P.A.R.I.S., France). The membrane was then washed 3 times with TBS-Tween 20 0.1% and incubated with Clarity Western Cl substrate (1705051, Biorad) and chemiluminescence was monitored using a Biorad ChemiDoc^TMXRS+.