Anti cdc25c c 20

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 was extracted in RIPA lysis buffer and identified by BCA assay. We separated denatured protein in SDS-polyacrylamide gel and transferred it to the Hybond membrane, which was blocked with 5% milk in TBST. For immunoblotting, the membrane was incubated for 1 h with mouse anti-Bax (Santa Cruz), rabbit anti-cdc25B (H-85, Santa Cruz), anti-cdc25C (C-20, Santa Cruz), anti-E-cadherin (Cell Signaling Technology), anti-N-cadherin (Cell Signaling Technology), anti-slug (Cell Signaling Technology), anti-twist1 (Cell Signaling Technology), or anti-GAPDH (Santa Cruz) antibody for 1 h in TBST at room temperature. Subsequently, these membranes were incubated with antimouse or antirabbit IgG conjugated to horseradish peroxidase (Dako) for 1 h at room temperature. Bands were visualized using ECL- Plus (Santa Cruz).