Foxm1 antibody

The paraffin-embedded tissue samples were collected from 20 women with primary epithelial ovarian cancer, stagesIIto IV, who had undergone initial surgery at the department of obstetrics and gynecology, Renji Hospital, School of Medicine, Shanghai Jiao Tong University between 2005–2008. The slides were deparaffinized, rehydrated and placed into citric acid buffer (pH 6.0, 0.1 M) for heating for 10 min. The endogenous peroxidase activity was then blocked by incubation with 3% H₂O₂ for 10 min. Afterwards, sections were incubated with blocking buffer (Beyotime, China) for 1 h and then incubated overnight at 4°C with FOXM1 antibody (1∶50, Santa Cruz). Following a 10-min incubation of biotinylated second antibody, the slides were again incubated with streptavidin-peroxidase under the same condition. The immunoreaction was then visualized by incubation with diaminobenzidine chromogen (DAB, Maixin-Bio, China) for 5 min. Finally, the slides were counterstained with hematoxylin, dehydrated, cleared and mounted. Negative controls were incubated in blocking buffer alone. These results were only considered if these control samples demonstrated a negative staining.

Chromatin immunoprecipitation (ChIP) assays were performed in EOC cells following the protocol provided by the manufacturer (Millipore, Bedford, MA). Briefly, after cross-linking with formaldehyde at 1% final concentration for 10 min at 37°C and the reaction was quenched by addition of glycine to a final concentration of 0.125 M. The cells were lysed in SDS buffer and the pellet was resuspended in nuclei lysis buffer and sonicated. Immunoprecipitation was carried out with FOXM1 antibody (Santa Cruz Biotechnology, Dallas, TX). The PCR primer sequences for DNA fragments as parts of the targeted promoters are provided in Supplementary Table S2.

Co-IP was performed using the Pierce Co-IP Kit from Thermo Scientific following the manufacturer’s protocol. Briefly, the FoxM1 antibody (Santa Cruz) was first immobilized for 2 h using AminoLink Plus Coupling Resin. The resin was then washed and incubated with the lysate of lung tissues overnight. A negative control that was provided with the IP kit to assess nonspecific binding received the same treatment as the Co-IP samples. Immunocomplexes were washed five times with washing buffer before being resolved by SDS-PAGE and immunoblotted with the indicated antibodies.

Western-blot analyses were performed as previously described [26 (link)]. The following antibodies were used: mouse monoclonal MELK antibody (Oncotherapy Science), and rabbit polyclonal FOXM1 antibody (Santa Cruz, Dallas, TX), mouse Cyclin B1 antibody (Santa Cruz), Caspase-3 and cleaved Caspase-3 (Cell Signaling, Danvers, MA) mouse monoclonal β-Actin (AC-15) (Sigma-Aldrich).

Western blot analysis to assess FOXM1 (1:500, Santa Cruz Biotechnology, Santa Cruz, CA) and GADPH (1:1000, Proteintech, China) expression was carried out as described previously [18 (link)]. GADPH primary antibody was purchased from Sigma (St. Louis, MO, USA). FOXM1 antibody was purchased from Santa Cruz Biotechnology, Inc. USA.

Chromatin immunoprecipitation (ChIP) assays were performed in lung adenocarcinoma cells following the protocol provided by the manufacturer (Millipore, Bedford, MA). Briefly, after cross-linking with formaldehyde at 1% final concentration for 10 min at 37°C and the reaction was quenched by addition of glycine to a final concentration of 0.125 M. The cells were lysed in SDS buffer and the pellet was resuspended in nuclei lysis buffer and sonicated. Immunoprecipitation was carried out with FOXM1 antibody (Santa Cruz Biotechnology, Dallas, TX). The PCR primer sequences for DNA fragments as parts of the targeted promoters are provided in Supplementary Table S2.