Peroxidase linked secondary antibodies

Western blotting was performed as described previously [28 (link)]. The protein-transferred membranes were incubated overnight at 4°C with primary antibodies for TrkB (1:200, sc-8316, Santa Cruz Biotechnology, Santa Cruz, CA, USA), p-TrkB (1:500, ab197072, Cambridge, MA, USA), BDNF (1:200, sc-546, Santa Cruz Biotechnology), MMP-2 (1:200, sc-10736, Santa Cruz Biotechnology), MMP-9 (1:200, sc-6840, Santa Cruz Biotechnology), E-cadherin (1:200, sc-7870, Santa Cruz Biotechnology), vimentin (1:200, sc-6260, Santa Cruz Biotechnology), SLUG (1:200, sc-15391, Santa Cruz Biotechnology), SNAIL (1:200, sc-10433, Santa Cruz Biotechnology), twist (1:200, sc-15393, Santa Cruz Biotechnology), VEGF-C (1:200, sc-7133, Santa Cruz Biotechnology), VEGF-D (1:200, sc-7603, Santa Cruz Biotechnology), p-MEK-1/2 (1:200, sc-7995, Santa Cruz Biotechnology), Erk1/2 (1:200, No9102 Cell signaling Technology), p-Akt1/2/3 (1:200, sc-101629, Santa Cruz Biotechnology), or Akt1/2/3 (1:200, sc-8312, Santa Cruz Biotechnology). Peroxidase-linked secondary antibodies (Amersham Biosciences, Piscataway, NJ, USA) were subsequently added and the membranes were further incubated for 1 h at room temperature. The antibodies for α-tubulin (1:1000, Sigma-Aldich, St. Louis, MO, USA) and β-actin (1:200, sc-47778, Santa Cruz Biotechnology) were used as protein loading controls.

Western blotting was performed as described previously 12 . Whole cell extraction was performed with M-PER reagents (Pierce Biotechnology, Rockford, IL) according to the manufacturer's instructions. Protein concentrations were determined with the Bio-Rad Protein Assay (Bio-Rad, Hercules, CA), and protein samples (50 μg) were separated by electrophoresis on a sodium dodecyl sulfate (SDS)-polyacrylamide gel and transferred to Protran nitrocellulose membranes (Whatman, Dassel, Germany). The protein-transferred membranes were incubated overnight at 4℃ with primary antibodies for FAM115C (1:500, GTX123264; GeneTex, Irvine, CA). Peroxidase-linked secondary antibodies (Amersham Biosciences, Piscataway, NJ) were subsequently added and the membranes were further incubated for 1 hr at room temperature. The antibodies for α-tubulin (1:1000; Sigma-Aldrich, St. Louis, MO) were used as protein loading controls.

Tumor cells were stimulated for 4 days with CM/CAF^TE4 or CM/CAF^OE19 (TE4^CM/CAF or OE19^CM/CAF) or cocultured with fibroblasts (TE4^DC or OE19^DC). In the coculture model, the tumor cells mixed with FEF3 cells were isolated using the same method as that used for flow cytometry. FEF3 cells were stimulated with CM from tumor cells (CM/TE4 or CM/OE19) for 2 days (CAF^TE4 or CAF^OE19). Primary antibodies against E-cadherin (Cell Signaling Technology), N-cadherin (Cell Signaling Technology), Vimentin (Cell Signaling Technology), αSMA (Abcam), and β-actin (Sigma) were used. Cells were washed, lysed in SDS buffer, and centrifuged. The supernatants were collected and subjected to WB. Proteins were electrophoretically transferred to Hybond-polyvinylidene difluoride transfer membranes (GE Healthcare Life Science) and incubated with a primary antibody, followed by peroxidase-linked secondary antibodies (Amersham Bioscience). An ECL Prime Western Blotting Detection Reagent (GE Healthcare UK Ltd.) was used to detect the peroxidase activity of the bound antibody.