Mouse monoclonal anti cyclin e

Cells were lysed in buffer consisting of 50 mM Tris-HCl pH 8, with 1% NP-40 (Igepal AC-630) 150 mM NaCl, 5 mM EDTA and fresh protease inhibitors. Protein concentrations were determined by colorimetric assay (Bio-Rad). Western blotting was performed using the following primary antibodies: mouse monoclonal anti-Tubulin (Santa Cruz Biotechnology), mouse monoclonal anti-Gapdh (Santa Cruz Biotechnology), rabbit polyclonal anti-p21 (Santa Cruz Biotechnology), rabbit polyclonal anti-Cyclin A (Santa Cruz Biotechnology), mouse monoclonal anti-Cyclin E (Santa Cruz Biotechnology), rabbit monoclonal anti-EGFR (Cell Signaling Tecnology, C74B9), rabbit polyclonal anti-N-Cadherin (Abcam). Secondary antibodies used were goat anti-mouse and goat anti-rabbit conjugated to horseradish peroxidase (Santa Cruz Biotechnology).

8–10% polyacrylamide gels were used for protein separation and detection. The gels were transferred to a nitrocellulose membrane, and antibody hybridization and electrochemiluminescence (ECL) were performed according to standard procedures. The primary antibodies used in this analysis were rabbit monoclonal anti-phosphorylated ATM 1:1,000 (Abcam), mouse monoclonal anti-ATM 1:10,000 (sigma), rabbit monoclonal anti-phosphorylated CHK1 1:200 (Cell Signaling), mouse monoclonal anti-CHK1 1:500 (Abcam), mouse monoclonal anti-cyclin E 1:200 (Santa Cruz Biotechnology), and rabbit polyclonal anti-beta actin 1:5,000 (Abcam). HRP-conjugated anti-rabbit and anti-mouse secondary antibodies were obtained from Jackson ImmunoResearch laboratories (West Grove, PA).

Alpha-MEM, EGF, bFGF, propidium iodide (PI), and RNAase A were purchased from Invitrogen (Hong Kong, China). HOE140, 5-Bromo-2′-deoxyuridine (BrdU), and mouse monoclonal anti-BrdU antibody were from Sigma-Aldrich (St. Louis, MO USA). Rabbit polyclonal anti-Akt, anti-pAkt(Ser473), anti-ERK1/2 and anti-pERK1/2 antibodies were from Cell Signaling (Danvers, MA, USA). Mouse monoclonal anti-BDKRB2 antibody was from Millipore (Merck Millipore, Germany). B2R siRNA, mouse monoclonal anti-cyclin D1, mouse monoclonal anti-cyclin E, HRP-conjugated goat anti-rabbit or rabbit anti-mouse IgG antibody were products of Santa-Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Transwell permeable support polycarbonate membrane and other cell culture flasks and plates were purchased from Corning Inc. (NY, USA).