The midguts were removed, washed in PBS buffer and homogenised in cold (4°C) in lysis buffer containing 20 mM Tris-HCL, 137 mM NaCl, 2 mM EDTA, 0.1% Triton-X 100 and 10% glycerol [ 105], supplemented with 0.1 – 0.2 mM PMSF (Sigma, St . Louis MO, USA) and a 200-fold dilution of peptidase inhibitor cocktail (Sigma).
For endogenous co-immunoprecipitation liver was extracted from a 3 mth old male mouse and homogenised in buffer B (Brinkmann polytron).
Briefly, snap-frozen tissue was weighed and homogenised in buffer containing 10 mM Hepes, 10 mM NaCl, 1 mM KH2PO4, 5 mM NaHCO3, 5 mM EDTA, 1 mM CaCl2, 0.5 mM MgCl2 and protease inhibitors (10x vol/weight).
Aliquots of muscle samples were weighed and homogenised in buffer (20 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, 100 mM KCl, 0.2 mM EDTA, 2 mM EGTA, 1 mM dithiothreitol, 50 mM NaF, 1 mM 3,3'-diaminobenzide tetrahydrochloride (DAB), 0.5 mM orthovanadate, and 50 mM glycine, pH 7.4) followed by centrifugation at 10,000 × g for 15 min at 4°C.
Cells were washed in cold PBS and homogenised in lysis buffer containing 50 mM Tris-Hcl pH 7, 100 mM NaCl, 50 mM NaF, 3 mM Na3VO4, protease inhibitor cocktail (Sigma-Aldrich) and 1% Nonidet P-40.
Cells were then pelleted, washed and homogenised in lysis buffer (10 mM Hepes, pH 7.9, 150 mM NaCl, 1 mM EDTA, 0.6% NP-40, and 0.5 mM PMSF) on ice.
For immunoprecipitation studies tissues and cells were harvested and homogenised in lysis buffer (150 mM NaCl, 10% Glycerol, 1 mM glycerophosphate, 1 mM sodium orthovanadate, 1 mM sodium fluoride).
For western blotting, embryos were manually dechorionated and homogenised in lysis buffer then processed as described above.
Tumours were surgically extracted at day 10 and homogenised in Net Buffer (100 m Tris, pH 7.4, 300 m NaCl, 10 m EDTA, 2%NP400).
Cells were washed with ice-cold PBS and homogenised in RIPA buffer by sonication as previously described (Nakanishi et al, 2006c).
Xenografts were removed and homogenised in lysis buffer (150 m M NaCl, 50 m M Tris, 1% NP40, 0.2% SDS, 1 m M PMSF, 10 μg ml−1 aprotinin, 10 μg ml−1 leupeptin and 1 m M sodium orthovanadate).
