Mouse monoclonal anti cyclin a2

Protein extraction and immunoblotting were performed as previously described [19 (link)]. The following antibodies were used: rabbit polyclonal anti-FOXF1 (Abcam #ab23194, 1:500), rabbit monoclonal anti-p21 (Cell Signaling Technology #2947, 1:2000), mouse monoclonal anti-cyclin A2 (Cell Signaling Technology #4546, 1:1000), rabbit polyclonal anti-cyclin B1 (Cell Signaling Technology #4138, 1:1000), rabbit polyclonal anti-cyclin E2 (Cell Signaling Technology #4132, 1:750), and mouse monoclonal anti-β-actin (Millipore #MAB1501, 1:10000).

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).