Anti cdc2 p34

The cells were treated with ATF₂₄-PEG-Lipo-β-E and/or DDP for the indicated times. The PVDF membranes were incubated with anti-cyclin B1 (1:1,000, Cell Signaling Technology, anti-Bcl-2 (1:1,000; Cell Signaling Technology), anti-Bax (1:1,000; Cell Signaling Technology), anti-cleaved PARP (1:1,000; Cell Signaling Technology), anti-Cdc25C (1:200; Santa Cruz Biotechnology, Dallas, TX, USA), anti-Cdc2 p34 (1:200; Santa Cruz Biotechnology), anti-cleaved caspase-3 (1:400; Abcam, Cambridge, UK), and anti-GAPDH (1:1,000; Cell Signaling Technology) primary antibodies at 4 °C overnight. The other steps were the same as described above³⁸ .

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.

RPMI medium 1640 (1x) + GlutaMAX was obtained from Gibco (Live Technologies) and Fetal Bovine Serum (FBS) from Biochrom AG (Berlin, Germany), and propidium iodide (PI), 4′-6-diamidino-2-phenylindole (DAPI), bicinchoninic acid solution, and gentamicin were obtained from Sigma-Aldrich Quimica, S.A. (Madrid, Spain). Metformin and the annexin V-FITC Apoptosis Detection Kit were purchased from Calbiochem (Darmstadt, Germany) and the XTT Cell Proliferation Kit II and RNase from Roche Molecular Biochemicals (Indianapolis, IN, USA). The Vybrant FAM caspase-3 and caspase-7 assay kit and DiOC₆(3) were obtained from Molecular Probes (Live Technologies).
The rabbit polyclonal anti-cyclin A (1 : 500, sc-751), anti-cyclin E (1 : 500, sc-481), anti-PKCε (1 : 200, sc-214), and anti-PKCδ (1 : 200, sc-213) antibodies and the mouse monoclonal anti-cyclin B1 (1 : 500, sc-245) and anti-cdc2 p34 (1 : 500, sc-54) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The rabbit polyclonal anti-LC3B (1 : 3000, ab51520) and the HRP-conjugated goat secondary polyclonal antibody against rabbit IgG-H&L (1 : 3000, ab6721) were purchased from Abcam (Cambridge, UK). Goat F(ab′)₂ anti-mouse IgG (H+L) was obtained from Southern Biotech (1032-05) and the rabbit anti-actin (1 : 200, A2066) antibody was from Sigma-Aldrich Quimica, S.A. (Madrid, Spain).