A11945

Total protein from tissue specimens and cells was extracted by sample buffer (62.5 mmol/L Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, and 5% 2-β-mercaptoethanol, Sigma-Aldrich, USA). The concentrations of protein were then determined using the BCA protein assay kit (Beyotime, China). Western blot analysis was performed as described 14 (link). Briefly, after being separated in SDS-PAGE gels, proteins were transferred to nitrocellulose membranes (Bio-Rad, USA). The membranes were then incubated with different primary antibodies at 4°C, overnight. Afterwards, respective secondary antibodies were applied at room temperature for 1hour. ECL development solution was manipulated for the visualization of the expression of different protein. The antibodies used are listed as follow: anti-HIF-1α (dilution at 1:1000, A11945, Abclonal, China), anti-FAP (dilution at 1:800, BM5121, Boster, China), anti-E-cadherin (dilution at 1:1000, 3195, CST, USA), anti-Twist (dilution at 1:1000, ab175430, Abcam, England), anti-Snail (dilution at 1:1000, ab53519, Abcam, England).

Western blot analyses were performed using previously described methods.14 (link) Target proteins were detected by incubating the membranes with the following primary antibodies: GAPDH (ab8245; Abcam, USA), α-SMA (ab32575; Abcam, USA), hypoxia-inducible factor-1α (HIF-1α) (A11945; ABclonal, USA), Bcl-2 (catalog no. 2872; CST, USA), Bax (catalog no. 2772; CST, USA), p53 (catalog no. 2524; CST, USA), α-Tubulin (ab4074; Abcam, USA), Histone H3 (BM4855; Boster, China) and β-Actin (ac004; ABclonal, USA). Nuclear and cytoplasmic proteins were extracted using a subcellular fractionation kit (P0027; Beyotime, Shanghai, China) according to the manufacturer’s protocol. The results were captured using an automatic fluorescence/chemiluminescence image analysis system (Tanon, Shanghai, China).