Tissue homogenate preparation
As previously described,23 1mL of 0.9% NaCl and
EDTA-free protease inhibitor (Roche, Basel, Switzerland) was added per
0.1g of frozen tissue. Tissue was disrupted using a bead mill
homogeniser (FastPrep-24 5G, MP Biomedicals, Santa Ana, United States)
at 6m/sec, 40 sec, over 4 cycles at 4oC, with 180 sec
between runs, in 2mL tubes with a single 3.175mm stainless steel bead
(Metal Bead Lysing Matrix 1/8″ Matrix S, MP Biomedicals, Santa Ana,
United States). Homogenates were centrifuged (10,000xg, 10 min,
4oC) (Heraeus Fresco 17 Centrifuge, Thermo Fisher
Scientific, Waltham, United States). 37 Supernatants
were aliquoted into microcentrifuge tubes (Eppendorf, Hamburg, Germany),
and stored at -80oC until analysis.