Anti cdc25a

The IGR-CaP1 cell line was maintained in RPMI1640 medium supplemented with 10% FBS. Docetaxel-resistant clones were selected by exposing cells to Docetaxel in a dose-escalation manner as described [33 (link)]. Surviving clones to low dose of Docetaxel were subsequently subjected to 5nM, 12nM, 25nM, 50nM, 100nM and 200nM of Docetaxel. Cells freely dividing in each dose of Docetaxel-containing media were considered resistant. Docetaxel (TAXOTERE^®) was kindly provided by Sanofi-Aventis (France). NSC663284 was purchased from Calbiochem; BI2536 and CHIR-124 were purchased from Selleckchem and were resuspended in DMSO. Anti-LZTS1 (C-20), and anti-CDC25C (C-20) were obtained from Santa-Cruz Biotechnology, anti-CDC25A, anti-CDC25B, from Cell Signalling, anti-GAPDH and anti-β-actin from Sigma.

Protein extracts were prepared by using RIPA lysis buffer with freshly added protease inhibitor cocktail (Sigma, St. Louis, MO, USA) [26 ], separated by SDS-PAGE gel and transferred onto a nitrocellulose membrane (Millipore, Bredford, USA). After blocking with 5% skim milk, the membrane was incubated with primary antibodies [anti-STK39, anti-MMP-2 and anti-MMP-9 (Abcam, Cambridge, MA, USA); anti-PCNA, anti-CDC25A, anti-Snail, anti-E-cadherin, anti-p38, anti-p-p38 and anti-GAPDH (Cell Signaling, Danvers, MA, USA); anti-Bcl-2 and anti-Bax (Santa Cruz Biotech., Santa Cruz, CA, USA)] overnight at 4°C and then incubated with corresponding HRP-conjugated secondary antibody (Beyotime, Shanghai, China) according to the manufacturers' instructions. Signals were detected with enhanced chemiluminescence system (Bio-Rad, Richmond, CA, USA).

Cells were rinsed with PBS, and then harvested, lyzed in lysis buffer (Applygen Technologies Inc. China) for 30 min on ice, centrifuged 12000 g for 20 min at 4 °C. Protein concentrations were measured by BCA assay. 30 µg of protein of each group was resolved by 10% SDS-PAGE, and further transferred to PVDF membrane. The membrane was blocked in 5% fat-free milk with TBST for 1 h at room temperature. The membrane was incubated with anti-γ-H2AX, anti-p21, anti-p-ATM, anti-ATM, anti-p-Chk2, anti-Chk2, anti-Cdc25A, anti-p-p53, anti-AKT, anti-active caspase-3 and anti-caspase-3 (Cell Signaling Technology, USA), anti-p53, anti-Bax, anti-Bcl-2, anti-Fas, anti-Fas L anti-α-tubulin (Santa Cruz, USA) antibodies in 5% milk TBST, at 4 °C overnight. After washed 3 times, the membrane were incubated with secondary antibodies for 1 h. Then, membranes were washed and subsequently developed using ECL (FujiFilm, Japan) reagent (Applygen Technologies Inc. China). All immnuoblot images were quantified by Gel-Pro analyzer4.0 software.