Briefly, cells were suspended in cell lysis buffer and homogenized on ice using a Dounce homogenizer (10 30 strokes) and centrifuged at 600×g for 10 min at 4°C.
The cord was placed in cold lysis buffer and homogenized on ice using a tissue homogenizer (Tissue Tearor 985370, Biospec Products, Bartlesville OK, USA).
Individual cerebral cortexes were weighed, suspended in 3 ml of phosphate buffer and homogenized on ice with a sonic dismembrator.
The wrist and ankle joints were snap-frozen in liquid nitrogen, ground into a fine powder by mortar and pestle, then lysed with protein lysis buffer (1% Triton X-100, 150 m M NaCl, 10 m M Tris-base, 1 m M EDTA, 1 m M EGTA, pH 7.4, protease inhibitor cocktail; 50 mg specimen per milliliter of lysis buffer), and homogenized on ice for 20 s.
Confluent cells in 100 mm dishes were rinsed once with PBS, lysed in 1 ml of ice-cold Hepes (250 mM) – Sucrose (10 mM) buffer (pH 7.4) and homogenized on ice using a Dounce tissue homogenizer.
After removal of RNAlater, samples were incubated in 600 µl RLT-β-mercaptoethanol buffer (1 100) for 10 min and homogenized on QIAshredder columns (Qiagen).
STR and VMB were removed rapidly and homogenized on ice with lysis buffer (Tris-HCl, pH 7.5, 20 mM; NaCl, 137 mM; NaF, 20 mM; sodium pyrophosphate, 1 mM; Na3VO4, 1 mM; Nonidet P-40,1%; glycerol, 10%; phenyl methyl sulphonyl fluoride, 1 mM; and leupeptin, 1 µg/ml).
The sliver of tissue was then placed in a sterile Eppendorf tube, and homogenized on ice in RIPA buffer containing protease inhibitors.
Cells were washed with PBS and homogenized on ice in lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 25% glycerol, 1% Triton X-100) supplemented with a cocktail of protease inhibitors and fresh PMSF (0.5 mM) and DTT (1 mM).
To assess translocation of β-catenin from cytosol to nucleus, cells were trypsinized and homogenized on ice in lysis buffer containing 0.1 mmol/l EGTA, 0.1 mmol/l EDTA, 10 mmol/l Hepes, 10 mmol/l KCl, protease and phosphatase inhibitors.
Frozen gastrocnemius muscle was directly immersed in cold RIPA lysis buffer and homogenized using an Ultra-Turrax homogenizer and then left on ice for 1 hr.
