Anti wee1

Cell homogenates were obtained in RIPA buffer (Pierce, Thermo Scientific), supplemented with 1 × EDTA-free complete protease inhibitor cocktail (Roche). Protein concentration was quantified by a modified Lowry assay (DC protein assay; Bio-Rad). 30 μg of protein were resolved in NuPAGE 4-12% Bis-Tris gels and transferred to iBlot Gel Transfer Stacks PVDF membranes (Life Technologies, Thermo Fisher Scientific). After blocking with Tris-buffered saline with Tween-20 containing 5% non-fat dry milk or 5% BSA for 1 h at room temperature, membranes were probed overnight at 4°C with the following antibodies: anti-PARP [1:2000, Cell Signaling #9542]; anti-CDC25A [1:1000, Santa Cruz #sc-7389]; anti-WEE1 [1:1000, Santa Cruz #sc-5285]; anti-CHK1 [1:2000, Santa Cruz #sc-8408]; anti-BCL2 [1:1000, Dako #M0887]; anti-AKT3 [1:1000, Upstate #05-780]; anti-VEGF [1:1000, Abcam #ab46154]; anti-Mouse IgG-Peroxidase antibody produced in rabbit (1:10.000, Sigma-Aldrich #A9044); anti-Rabbit IgG-Peroxidase antibody produced in goat (1:10.000, Sigma-Aldrich #A0545) and anti-Actin HRP [1:40.000, Santa Cruz #sc-1616]. Membranes were developed with SuperSignal Dura detection kit (Pierce) or EZ-ECL Chemiluminescence detection kit (Biological Industries, Kibbutz Beit-Haemek, Israel).

Cell culture media, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), hematoxylin and eosin were purchased from Sigma. Foetal bovine serum (FBS) and antibiotics were from Life Technologies and Immobilon P (PVDF) membranes from Millipore. OOS was provided by Catalysis, S.L. (Madrid, Spain). The Cytometric Bead Array Mouse Inflammation kit was purchased to BD biosciences. Other generic chemicals were purchased from Sigma Chemical Co., Roche Biochemicals or Merck.
The origin of the different antibodies used in the Western blotting analyses were as follows: the anti-GAPDH, anti-p21, anti-Wee1, anti-CDC2/CDK1, anti-pCDC2, anti-Cyclin B and anti-Cyclin D1 antibodies were purchased from Santa Cruz Biotechnology; the anti-BUBR1, anti-Rb, anti-Cyclin A, anti-Cyclin D3 and anti-Cyclin E from BD Biosciences; the anti-p27 and the anti-pRb^S780 from Cell Signaling technology; the anti-Calnexin from Stressgen and the anti-pHistone H3 from Millipore. The horseradish peroxidase-conjugated secondary antibodies were from Bio-Rad.

Cell lysate extractions were prepared with RIPA buffer 1% NP-40; 0.1% sodium dodecyl sulfate; 0.5% desoxycholate; 150 mM NaCl; 50 mM Tris, pH 7.5) and a protease inhibiter cocktail. 20 μg total protein of each lysate was resolved in SDS PAGE gels and electro-transferred to PVDF membranes, and then blocked in 5% skim milk in 0.05% Tween-20 with 1X PBS (PBST). Primary antibodies were incubated with the blots at a 1:1000 dilution in minimal volumes of 5% BSA (Bovine serum albumin) in PBST buffer for 1 hr at room temperature or over-night at 4 °C. Anti-mouse or anti-rabbit goat-HRP-conjugated secondary antibodies were incubated at a 1:5000 dilution in 5% BSA in PBST buffer for 1.5 hrs at room temperature. Antibodies used in this study were anti-WEE1, anti-Cdc2 p34, anti-phospho-Cdc2 p34 (Thr 14/Tyr15), anti-Cyclin B1, and anti-GAPDH that were purchased from Santa Cruz Biotechnology. Anti-caspase-3 and anti-PARP were obtained from Cell Signaling. Anti-phospho-histone H2A.X was purchased from Milipore Corpoation. Anti-mouse and anti-rabbit polyclonal immunoglobulins were purchased from Bethyl Laboratories. Membranes that were probed with primary antibodies and secondary antibodies were detected by ECL solution (Amersham Life Science) using a LAS-3000 (Fujifilm) detector, according to the manufacturer's directions.