Sc 15393

Immunoblotting was analyzed as previously described^{18 (link)}. Briefly, the cell lysates were resolved by SDS-PAGE. PVDF membranes with proteins were blocked and incubated for 30 min. Antibodies against RCP (1:1000, 12849S), Snail (1:1000, 3879S), N-cadherin (1:1000, 4061S), Zeb1 (1:1000, 3396S), p-EGFR (1:1000, 4407S), EGFR (1:1000, 2232 S), MT1-MMP (1:1000, 13130S) and nonphospho (NP) β-catenin (1:1000, 19807S) were purchased from Cell Signaling Inc. (Danvers, MA). Antibodies against GAPDH (1:3000, sc-47724), Twist (1:1000, sc-15393), β-catenin (1:1000, sc-7963), and β1 integrin (1:1000, sc-53711) were purchased from Santa Cruz Biotechnology. An antibody against E-cadherin (1:1000, 610182) was obtained from BD Biosciences (Franklin Lakes, NJ). The immunoreactive bands were exposed via ECL (Thermo Fisher Scientific Inc.) using an Amersham Imager 600 (GE Healthcare, Buckinghamshire, UK).

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.

Protein extracts were prepared by scraping the cells in the lysis buffer containing 100 mM KCl, 5 mM MgCl2, 20 mM Hepes-KOH (pH 7.8), 2 mM DTT, 25% NP-40, and a protease inhibitor cocktail (Roche). 20 μg of the whole protein extract was loaded onto 4–12% NuPAGE Bis-Tris Pre-Cast Gels and resolved using the NuPAGE System followed by iBlot transfer (ThermoFisher). Primary antibodies were: CPEB1 dilution 1:1000 (ab73287 Abcam) TWIST1 H-81, dilution 1:1000 (sc-15393, Santa Cruz Biotechnology), anti-c-Myc 9E10, dilution 1:1000 (11 667 149 001 ROCHE), anti-ZEB1 OTI3G6, dilution 1:1000 (TA802298 Origene), β-Actin (C4) HRP, dilution 1:2000 (sc-4778 HRP Santa Cruz Biotechnology).