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Western blot

WC Wenhu Chen
LW Lifang Wang
XL Xiaoyan Li
CZ Changan Zhao
LS Liang Shi
HZ Hongguang Zhao
CH Chen Huang
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Total protein was isolated by lysing cells with RIPA buffer (Beyotime, China) and was quantified with a BCA assay kit (Sangon, China). Total protein (50 μg) was separated by electrophoresis (10% SDS-PAGE) and transferred to a PVDF membrane (Millipore, USA). After treatment with a skim milk powder for 2 h at room temperature, the membrane was incubated overnight with primary antibody reactive with either; E-cadherin (14472,CST), Vimentin (5741,CST), N-cadherin (13116,CST), SOX4 (PA5-41442, Thermo Scientific), Snail (3879,CST), ZO-1 (13663,CST), TIMP-1 (8946,CST), TIMP-2 (5738,CST), or GAPDH (5174,CST) at 4 °C. The next day, secondary antibodies conjugated with horseradish peroxidase (HRP) were incubated with the membranes for 2 h. Protein signals were detected with ECL reagents (Sangon, China).

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