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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.

OS-RC-2 and 786-0 cells were transfected with pYR-ATP1A1 or pYR plasmids for 48 h, and cell pellets were collected. Cell pellets and grated tissues were dissolved with RIPA buffer (Cat.# P0013C, Beyotime, China) to extract proteins. 100 μg proteins were separated on 10% SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membrane (Cat.# IPVH00010, China). After blocking with 5% skim milk (OXOID), the PVDF membrane was incubated with primary antibodies overnight at 4 °C. The primary antibodies against ATP1A1 (1:200, ab2872, Abcam, UK), MEK1/2 (1:500, sc436, Santa Cruz, USA), p-MEK1/2(1:500, sc-81503, Santa Cruz, USA), ERK (1:500, sc-292838, Santa Cruz), p-ERK (1:500, sc-136521, Santa Cruz) β-actin (1:500, sc-1616, Santa Cruz, USA), and GAPDH (1:1000, sc-365062, Santa Cruz) were respectively diluted in TBST buffer (50 mM Tris–HCl, with 150 mM NaCl, 0.1% Tween-20, pH 7.4). Then the PVDF was incubated with the HRP-tagging secondary antibodies (ZSGB-BIO, China) at 37 °C for 1 h. Signals were finally detected with Western blot reagent ECL (Amersham Biosciences).