Sc 502

Protein extraction was performed using the Protein Extraction Buffer Invitrogen (Thermo Fischer Scientific, Waltham, MA, US). Twenty micrograms of protein were run on a 7%, 10%, or 12% SDS-PAGE gel, depending on the size of the protein, and transferred onto a nitrocellulose membrane (GE Healthcare) using a wet-blotting system (Bio Rad Laboratories) for 2 h at 100 V. Non-specific binding was blocked by incubating the membranes in 5% skimmed milk in TBS-Tween for 2 h. The blocked membranes were incubated overnight at 4 °C with the primary antibodies diluted in TBS/BSA 2% against FOXM1 (1:500, sc-502, Santa Cruz), XIAP (1:1000, 2045S, Cell Signaling), c-IAP 1 (1:2000, AF8181, R&D Systems), survivin (1:1000, 2808S, Cell Signaling), Mcl-1 (1:100, #4572, Cell Signaling) and Hsc70 (1:1000, sc-7298, Santa Cruz). After overnight probing with primary antibodies, the membranes were incubated with secondary antibodies anti-mouse (1:40000, GE Healthcare), anti-rabbit (1:40000, GE Healthcare), and anti-goat (1:2000, NB7362, Novus Technologies; Littleton, CO, USA) for 1 h at room temperature. Protein band signals were developed using the Clarity Max™ substrate (Western ECL Substrate-BioRad Laboratories, Hercules, CA, USA) and detected using the C-DiGit blot scanner (Li-cor Biociences, Lincoln, NE, USA).

First, 3-μm-thick CRC tissue sections were deparaffinized, rehydrated, and treated with 3% H₂O₂ to block endogenous peroxidase activity. After the sections were pretreated for antigen retrieval by microwaving them in ethylenediamine tetraacetic acid (EDTA) (pH 9.0) for 25 min, they were rinsed in phosphate-buffered saline (PBS) and incubated with various primary antibodies overnight at 4 °C in a humidified chamber. The next morning, the slides were rinsed with PBS and then incubated for 40 min at 37 °C with the appropriate biotinylated immunoglobulins (Zhongshan Biotechnology, China) before visualizing the immunoreactivity using a Polink-2 HRP DAB Detection kit (Zhongshan Biotechnology, China) following the manufacturer’s protocol. Negative controls were performed in each case by replacing the primary antibody with normal IgG. The following primary antibodies were used: anti-Gli1 (Abcam, ab92611, diluted 1:100) and anti-FoxM1 (Santa Cruz, SC-502, diluted 1:300). An FSX100 microscope equipped with a digital camera system (Olympus, Japan) was used to obtain the immunohistochemistry images.

Primary Antibody used were: Alix(3A9) (1:1000 dilution, mouse monoclonal, mAb#2171, Cell Signalling), CD9 (1:200, rabbit monoclonal, ab92726, Abcam), CD63 (H-193) (1:1000, rabbit polyclonal, sc-15,363, Santa Cruz), CEP55 (1:10000, rabbit monoclonal, ab170414, Abcam), Calnexin (1:1000, rabbit polyclonal, ab22595, Abcam), Glypican 1 (1:500, rabbit polyclonal, ab55971, Abcam), FOXM1 (1:500, rabbit polyclonal, sc-502, Santa Cruz), GAPDH (1:10000, mouse monoclonal, ab8245, Abcam), HSC70(B-6) (1:10000, mouse monoclonal, SC-7298, Santa Cruz). Secondary antibody used were: Goat anti Rabbit IgG (1:1000; AP#132P, Millipore), Goat Anti-Mouse IgG (1:10000, A0168, Sigma). Additional information on immunoblotting methodology can be found in (Additional file 1).