Anti β actin

Total proteins were isolated by Total Extraction Kit (KGP2100; Nanjing KeyGen Biotech Co., Ltd., Nanjing, China) following the manufacturer's protocols. Protein concentration was determined by the BCA reagent kit (KGPBCA; Nanjing KeyGen Biotech Co., Ltd.). Equal amounts of protein (60 μg) were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes, which were blocked in 5% non-fat milk, and then incubated with the primary antibodies against anti-HIF-1α (1:1,000; ProteinTech Group, Inc.), anti-TGF-β (1:500; Abcam, USA), anti-Smad4 (1:1,000; ProteinTech Group, Inc.), anti-LOX (1:2,000; Abcam, USA), anti-E-cadherin (1:200; Cell Signaling Technology, Danvers, MA, USA) and anti-β-actin (1:1,000; Elabscience Biotechnology Co., Ltd.). The samples were incubated with the antibodies overnight at 4°C. Subsequently, the membrane was incubated with horseradish peroxidase-conjugated secondary antibodies (1:6,000; OriGene, Beijing, China) at room temperature for 1 h. An enhanced chemiluminescence reagent (ECL kit, KGP1121; Nanjing KeyGen Biotech Co., Ltd.) was applied as a chromogenic substrate for 1 min, and then visualized with an Amersham Imager 600 instrument (GE Healthcare Life Sciences). Grayscale analysis was performed with ImageJ software.