Phosstop inhibitor cocktail tablets

Rat tissues were homogenized in RIPA buffer (Thermo Scientific, Rockford, IL, USA) with protease (complete, Mini, EDTA-free) and phophatase (PhosSTOP) inhibitor cocktail tablets (Roche, Indianapolis, IN, USA). A similar homogenization buffer [35 (link)] was used for human samples. Homogenates were sonicated, incubated on ice for 15–30 min and then centrifuged at 13000 × g for 10–20 min at 4°C to precipitate insoluble debris. The supernatants were collected and protein concentrations were determined using the BCA protein assay kit (Thermo Scientific, Rockford, IL, USA) or the DC™ protein assay (Bio-Rad Laboratories, Mississauga, Ontario, Canada) as described by the manufacturer. Rat tissue lysates were pooled in order to handle a large number of samples that exceeded the lane capacity of a gel, while human homogenates were not pooled. Sample protein concentrations were adjusted to 4 μg/μl (rat) or 1.5-3.5 μg/μl (human) for Western blotting.

Homogenization of tissue took place in 300 μL of lysis buffer [0.15 NaCl, 1% TX-100, 10% glycerol, 1 mM EDTA, 50 mM TRIS pH = 7.4] containing a phosphatase and protease inhibitor cocktail [Complete ULTRA Protease Inhibitor Cocktail Tablets (Cat# 5892970001) and PhosSTOP Inhibitor Cocktail Tablets (Cat# 4906837001) respectively; Roche]. Centrifugation of homogenates took place at 12,200 rpm for 20 min at 4°C and supernatants were collected for quantification of protein content using the Pierce BCA protein assay kit (ThermoFisher, Cat# 23225), following manufacturer’s instructions.