Rabbit α ha

Supernatants were precleared for 1 h at 4°C with 75 μl of 50% Sepharose bead slurry (Amersham) and 1.5 μg of species-specific IgG (Sigma, 1 mg/ml). Protein G sepharose bead slurry and mouse IgG were used for the samples immunoprecipitated with mouse α Myc (9B11; Cell Signalling Technology); protein A sepharose bead slurry and rabbit IgG for the samples immunoprecipitated with the rabbit α HA (Cell Signalling Technology) antibody. Samples were then spun at 13,000 rpm for 5 min and the resulting supernatants were incubated overnight at 4°C respectively with mouse α Myc antibody (2 μl/1.5 mg proteins) or rabbit α HA (1:50) antibodies. The following day, the α Myc samples were incubated with 75 μl of 50% protein G sepharose beads and the α HA samples with 75 μl of 50% protein A sepharose beads for a further 3 h at 4°C. Samples were centrifuged at 3,000 rpm for 5 min at 4°C and the recovered pellets were washed three times in RIPA buffer [150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris pH = 8.0 and protease inhibitors (Roche)]. Samples were eluted with sample buffer containing DTT and denatured by boiling for 5 min. Co-immunoprecipitation samples (IP) were run together with the input (IN) and the supernatant (S) resulting from the immunoprecipitation, transferred as described above and blotted with mouse α Myc and rabbit α HA as described above.