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The cellular secretion level of STC2 in cell conditioned media from NCM460, HT29 and EMT cells, as well as human serum STC2 was measured by the enzyme-linked immunosorbent assay (ELISA). The ELISA procedures were mainly followed our previous methods [15 (link)]. The CM or serum samples were added into a 96-well plate, which was pre-coated with anti-STC2 antibodies (sc-14350, Santa Cruz, USA), to incubate for 1 h at 37°C. After washing 5 times with PBST (3.2 mM Na₂HPO₄, 0.5 mM KH₂PO₄, 1.3 mM KCl, 135 mM NaCl, 0.05% Tween 20, pH 7.4), the secondary antibody was added into the plate to incubate 1 h at 37°C. Finally then reactions were stopped with 1N HCl, and ELISA plates were detected using an ELISA Reader (Multiskan Mk3, thermo) at 450 nm, with correction at 570 nm.

The expression of STC2 and EMT markers was observed in cultured EMT cells by an immunofluorescence microscopy. Cells grown on coverslips, which were performed in a 96-well plate, were fixed with paraformaldehyde for 30 min. After fixation, cells were washed with TBST twice and blocked in 1.5% BSA/TBST for 1 h at room temperature. Cells were respectively incubated with primary antibodies against STC2 (1:500, sc-14350, Santa Cruz), vimentin (1:500, sc373717, Santa Cruz), E-cadherin (1:500, sc-31021, Santa Cruz), N-cadherin (1:500, sc-31031, Santa Cruz) and twist (1:500, sc-81417, Santa Cruz) overnight at 4°C. Then cells were incubated with TRITC-conjugated secondary antibodies (ZF-0313, ZSGB-BIO) and FITC-conjugated ones (ZF-0314, ZSGB-BIO) for 1 h and stained with DAPI for 5 min. The images were viewed and recorded by Olympus BX40 and SPOT Flex (Diagnostic Instruments, Version 4.5).
To block ERK, PI3K signaling, 10 μM ERK inhibitor U0126 (#S1901, Beyotime) or 10 μM PI3K inhibitor LY294002 (#9901, Cell Signaling Technology) was respectively incubated with NCM460, EMT cells for 24 h to directly observe expression strength of STC2 and EMT markers. Other procedures were same as the above.

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.