Quantitative Western Blot Analysis

The method for cell lysis and Western blotting is described elsewhere [23 (link)]. The primary antibodies used were mouse anti-Mad2 (Abcam, Cambridge, UK, ab10691, 1:500 or 1:1000), mouse anti-securin (Abcam, ab3305, 1:250), mouse anti-cyclin B1 (BD Biosciences, San Jose, CA, USA, 554178, 1:500), mouse anti-GAPDH (Advanced ImmunoChemical Inc., Long Beach, USA, or HyTest Ltd, Turku, Finland, mAb 6C5, 1:30 000-50 000), and mouse anti-E2F1 (Santa Cruz, Dallas, TX, USA, sc-251, 1:500). Secondary antibodies were Alexa Fluor^® anti-mouse 680 (Invitrogen), IR Dye^® conjugated anti-mouse 800 (Rockland Immunochemicals Inc., Gilbertsville, PA, USA) and HRP-linked anti-mouse IgG (Cell Signaling Technology). Secondary antibodies were used as 1:5000 dilutions with 1 h incubation at RT. The signal measurement and the quantitative analysis were done using a two channel Odyssey Infrared Imaging System (LI-COR Biotechnology) or ECL detection system and ImageQuant LAS4000 CCD camera (Fujifilm, GE Healthcare).