Tissue samples homogenization, protein extraction, denaturing polyacrylamide gel electrophoresis (SDS-PAGE), and WB was performed as previously described [27 (link)]. The membranes were subjected to blocking by using Tris buffered saline (TBS: 10 mM Tris-HCl, pH 7.4, 165 mM NaCl) added with 0.1% Tween 20 (TTBS) and 5% non-fat dry milk, at room temperature for 1 h. The anti-HIF antibody (#ab114977, Abcam) at 1:1000 dilution was incubated overnight at 4 °C.
Following four washing steps of 10 min in TTBS, donkey anti-rabbit secondary antibody conjugated with peroxidase was employed at 1:2000 dilution for 1 h at room temperature. After additional washing steps, bound antibody was visualized by enzyme chemiluminescence with Clarity ™ Western ECL Blotting Substrate (Bio-Rad Laboratories, Milano, Italy). The blots were stripped and reprobed for β-actin (CP01, Calbiochem, San Diego, CA, USA) (1:500) as a loading control in order to perform normalization. Protein quantization and normalization were performed as reported in a previous study [27 (link)].
Following four washing steps of 10 min in TTBS, donkey anti-rabbit secondary antibody conjugated with peroxidase was employed at 1:2000 dilution for 1 h at room temperature. After additional washing steps, bound antibody was visualized by enzyme chemiluminescence with Clarity ™ Western ECL Blotting Substrate (Bio-Rad Laboratories, Milano, Italy). The blots were stripped and reprobed for β-actin (CP01, Calbiochem, San Diego, CA, USA) (1:500) as a loading control in order to perform normalization. Protein quantization and normalization were performed as reported in a previous study [27 (link)].
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