Immobilion p membrane

Frozen tumor samples were homogenized and cell membranes were prepared by hypotonic lysis and high-speed centrifugation, as previously described [55 (link)]. Tumor membrane preparations, or cultured cells, were lysed with Laemmli buffer and proteins were separated by gel electrophoresis before their transfer onto Immobilion P membranes (Millipore, Bedford, MA, USA) for immunoblotting. Immune complexes were detected by enhanced chemiluminescence (GE Healthcare Amersham-Fisher Scientific, Waltham, MA, USA) and quantified using the GeneGnome Bio Imaging System (Syngene, Frederick, MD, USA). Immunoprecipitation was performed as described previously [56 (link)].

For immunoblotting, cells were seeded in 24 or 96 well plates (Corning) then later harvested by trypsinisation, centrifuged, washed and lysed in SDS buffer (0.35 M Tris pH 6.8, 0.1 g/ml sodium dodecyl sulfate, 93 mg/ml dithiothreitol, 30% glycerol, 50 μg/ml bromophenol blue). Proteins were resolved by SDS-PAGE then electro-blotted onto Immobilion-P membranes (Millipore). Membranes were blocked in 5% dried milk in TBST (50 mM Tris, pH 7.6, 150 mM NaCl, 0.1% Tween-20) then incubated overnight at 4°C with the following primary antibodies diluted in milk: rabbit anti-Mcl-1 (Santa-Cruz), sheep anti-Tao1 [60 (link)], mouse anti-Cyclin B1 (Millipore), rabbit anti-Bcl-xL (Cell Signalling), sheep anti-Bub3 (Holland and Taylor, unpublished), rabbit anti-FBW7 (Bethyl), mouse anti-Bak (Calbiochem), rabbit anti-Bax (Santa-Cruz), mouse anti-myc tag (4A6, Millipore), rabbit anti-GFP (Cell Signalling). Following TBST washes, blots were incubated with appropriate horseradish-peroxidase-conjugated secondary antibodies (Zymed). Bound secondaries were then detected by addition of EZ-ECL Chemiluminescence Reagent (Biological Industries) or Luminata™ Forte Western HRP Substrate (Millipore) and imaged using a Biospectrum 500 imaging system (UVP).

Cells were washed with cold PBS and homogenized in lysis buffer (50 mM Tris pH 7.6, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA and 1% SDS). Samples were heated at 95 °C for 15 min and sonicated. Protein quantification was performed with the DC^TM Protein Assay (Bio-Rad, Hercules, CA, USA; ID 5000112), and protein loading buffer (50 mM Tris-HCl pH 6.8, 2% SDS, 0.1% bromophenol blue, 10 % glycerol, 150 mM β-mercaptoethanol) was added. Samples were boiled at 95 °C and cellular debris was cleared with centrifugation. Proteins were resolved using SDS-PAGE and transferred to 0.45 µm pore size Immobilion-P membranes (Millipore, Burlington, MA, USA; ID IPVH00010). For immunoblotting, membranes were hydrated in methanol, washed in TTBS (20 mM Tris-HCl pH 7.5, 150 mM NaCl and 0.1% Tween 20) buffer and blocked with 5% non-fat dry milk in TTBS. Membranes were incubated with the appropriate dilution of the primary antibodies (Table 2) in 0.4% BSA or 2.5% non-fat-dry milk for 1 h, washed and incubated with 1:10,000 dilution of secondary antibodies coupled to horseradish peroxidase in 0.4% BSA TTBS for 1 h. Proteins were detected by carrying out enhanced chemiluminescence (Amersham^TM ECL^TM Select Western Blotting Detection Reagent, GE Healthcare, Chicago, IL, USA; ID GERPN2235).