Mouse monoclonal anti cyclin b1

Cell lysates were prepared using radioimmunoprecipitation assay (RIPA) lysis buffer (P0013E; Beyotime, Shanghai, China) supplemented with protease inhibitor cocktail (469,313,200; Roche, Basle, Switzerland). Immunoprecipitation and western blot analysis were carried out as described previously (Liu et al. 2009 ), with ISL1 for Co-IP (H00003670-M05, Abnova, Taipei, China) and ISL1 for western blotting (ab109517, Abcam). Monoclonal anti-CDK1 (#9116), GAPDH (#2118), rabbit polyclonal anti-CDK1-T161P (#9114), and mouse monoclonal anti-cyclin B1 (#4135) were purchased from Cell Signaling Technology (Danvers, MA, USA). Mouse monoclonal anti-phosphoserine (05-1000) was obtained from Merck Millipore (Millipore Corp, Billerica, MA, USA). Rabbit polyclonal antibody against the phosphorylation site of ISL1 at Ser 269 (ISL1-S269) (1:1,000) was produced with the synthetic phosphopeptide MTGTPMVAA[Sp]PERHDGGLQ. (Absin Bioscience Inc., Shanghai, China)

The cells were lysed in radio immunoprecipitation assay buffer (Beyotime, Shanghai, China) supplemented with a protease inhibitor cocktail (Roche, Basel, Switzerland). Equal amounts of proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5% non-fat milk in Tris-buffered saline containing Tween 20 for 1 h and incubated with primary antibodies at 4° C overnight. The primary antibodies were as follows: mouse polyclonal anti-NFAT5 (Abcam, Cambridge, UK), mouse monoclonal anti-cyclin A2, mouse monoclonal anti-cyclin B1, rabbit monoclonal anti-MMP-2, rabbit monoclonal anti-MMP-9, and mouse monoclonal anti-β-actin (all from Cell Signaling Technology, Beverly, MA, USA). The membranes were then incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (Sigma) for 60 min. The protein bands were visualized using an enhanced chemiluminescence kit (Santa Cruz, Dallas, TX, USA) and exposed to X-ray film (Kodak, Fujian, China).