GST-BCR was transfected into HEK 293 cells and was immunoprecipitated from 2 mg of cellular lysate using different amounts (as indicated) of anti-BCR (1207 – 1227), pre- immune serum or GSH-Sepharose. The immunoprecipitates were subjected to SDS-Page transferred to nitrocellulose and immunoblotted with anti-GST. Binding of the primary antibody was detected using rabbit peroxidase conjugated anti- sheep IgG antibody (1 in 10, 000 dilution, Pierce) followed by enhanced chemiluminescence (ECL, Amersham).
Endogenous BCR was immunoprecitated from 2 mg of HEK 293 and mouse Swiss 3T3 cellular lysates using 2 ug of anti-BCR (1207 – 1227). The immunoprecipitates were subjected to SDS-Page transferred to nitrocellulose and immunoblotted with anti-BCR [cell signalling, cat no: 3902]. Detection of the primary antibody was performed according to manufacturer’s instructions.