Western Blot Analysis of EMT Markers

Several target proteins were detected their expression by Western blot. Proteins were separated on a 12% SDS-PAGE gel and transferred onto a PVDF membrane at 100 V for 1 h. The specific primary antibodies, including vimentin (1:500, sc373717, Santa Cruz), E-cadherin (1:500, sc-31021, Santa Cruz), N-cadherin (1:500, sc-31031, Santa Cruz), twist (1:500, sc-81417, Santa Cruz), STC2 (1:500, sc-14350, Santa Cruz), ERK (1:500, sc-292838, Santa Cruz), MEK (1:500, sc-436, Santa Cruz), p-ERK (1:500, sc- 136521, Santa Cruz), p-MEK (1:1000, 9121, Cell signaling), Akt (1:1000, 4961, Cell signaling) and phospho Akt (1:1000, 2118–1, Epitomics), were diluted in TBST buffer (50 mM Tris–HCl, with 150 mM NaCl, 0.1% Tween-20, pH 7.4) to incubate PVDF membrane at 4°C overnight. The corresponding secondary antibodies, conjugated horseradish peroxidase, were subsequently incubated with the PVDF membrane at 37°C for 1 h. Signal detection was performed with HRP substrates (WBLUR0100, Millipore). The detection of GAPDH against its antibody (1:1000, sc- 365062, Santa Cruz) was taken as a control.
For serum STC2 detection, the high abundant serum albumin and IgG was removed by using a reagent kit (ProteoExtract Albumin/IgG Removal Kit, 122642, Calbiochem, San Diego, CA) [38 (link)]. Reversible Ponceau staining was used as a loading control.