Anti cyclin b antibody

Western blotting was performed as described previously [66 (link)]. 20 fly heads were homogenized and underwent SDS-PAGE before transferred to PVDF membranes (Immobilon-P, Millipore). The membrane was blocked with 5% milk and incubated with anti-cyclin B antibody (Santa Cruz Biotechnology sc-15872 which has been proved to be specific and sensitive for western blot [21 (link)]), or anti-tubulin monoclonal antibody (1:5000, Sigma) overnight at 4°C. After incubation with HRP conjugated donkey anti-goat or goat-anti-mouse secondary antibody and reacted with SuperSignal West Pico chemiluminescent substrate (Pierce), tubulin and cyclin B expression measured by exposure to X-Omat BT film (Eastman Kodak)

Cells were lysed in lysis buffer containing 3.3% SDS, 1.67M urea and 4.4% 2-β-mercaptoethanol and then running onto 10% SDS-polyacrylamide gels. The protein bands were electrophoretically transferred to Hybond-C super membranes (Amersham), and probed with primary antibodies and followed with a horseradish peroxidase-conjugated secondary antibody. Finally, the signals were detected using an enhanced chemiluminescence substrate (PerkinElmer) and exposure to X-ray film (Fujifilm). Anti-BRLF1 antibody was obtained from Argene. Anti-phospho-Erk1/2 (Thr202/Tyr201), anti-Erk1/2 and anti-β-actin antibodies were purchased from Cell Signaling Technology. Anti-cyclin B antibody was obtained from Santa Cruz. Anti-securin and anti-GAPDH antibodies were obtained respectively from Abcam and GeneTex.