Cdc25c

The A2780 cell line was purchased from American Type Culture Collection (ATCC), and SAHA was purchased from Selleckchem (Houston, TX, USA). Primary antibodies against Ac-H2A, Ac-H2B, Ac-H3, Ac-H4, HDAC2, HDAC3, HDAC4, DNMT3A, PRMT1, SUV39H1, MDMX, p53, p21^WAF1/CIP1, p27^Kip1, AURKB, CDC25C, GADD45A (1:1000) and Alexa Fluor 488 dye were purchased from Cell Signaling Technology (Danvers, MA, USA). The MDM2 antibody (1:500) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). β-actin (1:5000), secondary antibodies (anti-rabbit, anti-mouse), horseradish peroxidase (HRP) conjugate, and dimethyl sulfoxide (DMSO) were purchased from Sigma Aldrich (St. Louis, MO, USA). Nitrocellulose membrane (NC) (0.45 µm) was purchased from Amersham (GE Healthcare Life Sciences, Marlborough, MA, USA) and KPL LumiGlo Reserve chemiluminescent substrate was purchased from SeraCare Life Sciences (Milford, MA, USA).

Foetal bovine serum (FBS) and Dulbecco's modified Eagle's medium (DMEM) were obtained from Thermo Fisher Scientific (Waltham, MA) and 4′,6-diamidino-2-phenylindole (DAPI) was from Sigma-Aldrich (Seelze, Germany). ER-tracker Red was obtained from Thermo Fisher Scientific (Waltham, MA). Mouse monoclonal antibodies (mAbs) against PERK, phosphorylated-eIF2a, CHOP, XBP-1s, cyclin A2 and Cdc25C were obtained from Cell Signaling Technology (Danvers, MA); and anti-β-actin and CDK1 were from Boster (Pleasanton, CA). Salubrinal was purchased from Calbiochem (San Diego, CA).

CA4 is purchased from Santacruz. All other chemical and reagents were obtained from Fisher Scientific Company. Antibodies to cleaved PARP, cleaved caspase, cyclin B1, aurora kinase A, CDK1 and CDC25C, COX-2, NF-κβ, E-Cadherin and Snail proteins were purchased from Cell Signal Technology.

Western blot analysis was performed using standard procedures for whole-cell extracts from cell lines. Antibodies used include Chk2 (1:5000, Becton Dickinson and Millipore), SIRT1 (1:2000, Millipore), acetyl-lysine (1:1000), phospho-CHK2-T68 (1:1000), phospho-CHK2-Thr387 (1:1000), phospho-p53-Ser20 (1:1000), acetyl-p53-Thr382 (1:1000), phospho-histone H2A.X (Ser139) (1:1000), phospho-ATM-Ser1981 (1:1000), ATM (1:1000), phospho-histone H3-S10 (1:1000), p-CDC25C (ser216) (1:1000), CDC25C (1:1000), cleaved PARP-1 (1:1000) and cleaved caspase-3 (1:1000) (Cell Signaling Technology), phospho-CHK2-Thr432 (1:500, Invitrogen), P53 (Do-1, 1:1000, Santa Cruz Biotechnology), FLAG (clone M2) (1:2000), α-tubulin (1:5000, Sigma), and β-actin (1:5000, Sigma). For immunoprecipitation analysis, cell lysates (1–4 mg) after preclearing were mixed with antibodies (2 μg) at 4 °C overnight followed by the addition of 30 μl of protein-G (for mouse antibodies)- or protein-A (for rabbit antibodies)-coupled sepharose beads (GE) for 3 h at 4 °C. Immune complexes were washed three times with lysis buffer [50 mM Tris (pH 7.4), 1% Triton X-100, 0.5% Nonidet P-40, 150 mM NaCl, protease, phosphatase inhibitor mixture (Sigma)]. After boiling in 2× loading buffer, samples were subjected to SDS-PAGE, and then scanned using ECL.

Rat monoclonal anti-BrdU (#OBT0030; AbDSerotec, Raleigh, NC), rabbit polyclonal anti-cytoskeletal actin (β-actin) (#A300-491A; Bethyl Laboratories, Inc., Montgomery, TX) and mouse monoclonal anti-Ki67 (NCL-Ki67-MM1; Novocastra/Leica Microsystems, Inc., New Castle, UK), rabbit polyclonal to p85 fragment of PARP (#G734A, Promega, Madison, WI) and cyclin B (BD #61029; BD Biosciences, San Jose, CA) were procured from their respective manufacturers. Rabbit polyclonal to cleaved caspase-3 (#9661), p21^Cip1/WAF1 (#CS2947), Cdc25C (#CS4648), p-Cdc25C (#CS9528), Cdc2 (#CS9112) and p-Cdc2 (#CS9111) were purchased from Cell Signaling Technology, Inc., Danvers, MA. Antibodies for p53 (#SC-6243), p27^kip21 (#SC528), p19 (#SC-71810), cyclin E (#SC481), Cdk2 (#SC6248) and Cdk4 (#SC23896) were all from Santa Cruz Biotechnology Inc., CA. Peroxidase-conjugated secondary (H&L chain-specific) antibodies for Western blots, goat anti-mouse IgG (#401253) and goat anti-rabbit IgG (#401315) were purchased from Calbiochem-Novabiochem Corp., CA. Secondary antibodies for immunocytochemical analyses, Cy3-AffiniPure donkey anti-rat IgG (#712-165-153) and Cy3-AffiniPure goat anti-mouse IgG (#115-165-003) were procured from Jackson Immuno Research Laboratories, Inc., West Grove, PA.

Western blot and protein detection was performed as previously described [43 (link)]. The following primary antibodies were used: RB, phospho-RB^S807/S811, CDC25C, phospho-AKT^S473, phospho-cyclin E1^T62, phospho-ERK1/2^T202/Y204, ERBB2, phospho-ERBB2^Y1221/1222 and MYC (Cell Signaling, Danvers, MA); BRCA1, p53, cyclin E, CDK1, CDK2, ETV4, ETV5 and HRAS (Santa Cruz Biotechnology, Santa Cruz, CA); Actin (Abcam, Cambridge, MA); GAPDH (Fitzgerald, Acton, MA); β-Actin (Sigma-Aldrich); PAX8 (Epitomics, Burlingame, CA);