Protein Extraction and Western Blotting

Protein extraction and blotting were performed as previously described [42 (link)]. After determination of protein concentration by a Protein Determination Kit (Cayman), equal amounts (50~100 μg) of protein samples were size fractionated using SDS-PAGE, electrotransferred onto PVDF membrane (Bio-Rad) and hybridized with antibodies. Antibodies used were for S1P₁ (TA311878, OriGene), Syndecan-1 (sc-390791, Santa Cruz), p-AKT (sc-33437, Santa Cruz), AKT (sc-8312, Santa Cruz), p-ERK1/2 (#9101, Cell Signaling), ERK1/2 (#4695, Cell Signaling), TGF-β1 (sc-52893, Santa Cruz), E-cadherin (sc-1500, Santa Cruz), Vimentin (sc-58899, Santa Cruz), Snail (#3879, Cell Signaling), and GAPDH (sc-137179, Santa Cruz). A 1:1000 dilution of those antibodies was used for detection. Densitometric quantification of bands was analyzed by the ImageJ software (version 1.50, NIH, USA).

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Zeng Y., Yao X., Chen L., Yan Z., Liu J., Zhang Y., Feng T., Wu J, & Liu X. (2016). Sphingosine-1-phosphate induced epithelial-mesenchymal transition of hepatocellular carcinoma via an MMP-7/syndecan-1/TGF-β autocrine loop. Oncotarget, 7(39), 63324-63337.

Publication 2016

Protein Determination Kit PVDF membrane Syndecan-1 p-AKT p-ERK1/2 ERK1/2 TGF-β1 E-cadherin Vimentin Snail GAPDH

A protein Antibodies Cayman Densitometric Dilution E cadherin Erk1 Gapdh Membrane Protein Pvdf S1p1 Sds page Snail Syndecan 1 Tgf β1 Vimentin

Corresponding Organization :

Other organizations : Sichuan University, Sun Yat-sen University, Sun Yat-sen University Cancer Center

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Variable analysis

independent variables

Protein extraction and blotting protocol

dependent variables

Protein concentration
Protein expression levels of S1P1, Syndecan-1, p-AKT, AKT, p-ERK1/2, ERK1/2, TGF-β1, E-cadherin, Vimentin, and Snail

control variables

Protein loading amount (50~100 μg)
GAPDH as a loading control

controls

No positive or negative controls were explicitly mentioned in the provided information.

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