After treatment, cells were washed with cold PBS, harvested, and lysed in radioimmunoprecipitation assay (RIPA) buffer containing protease and phosphatase inhibitors as described previously . The lysates were centrifuged at 14,000 rpm for 20 min at 4 °C, and the protein concentration of the supernatant was determined using a Bradford protein assay kit (Bio-Rad Laboratories Inc., Hercules, CA, USA) according to the manufacturer’s instructions. Protein samples were mixed with 4× Laemmli sample buffer (62.5 mM Tris-HCl (pH 6.8), 10% glycerol, 1% SDS, and 0.005% bromophenol blue), and equal amounts (30 μg/well) of protein were then subjected to 10 or 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at 90–100 V for 2 h. Next, proteins in a separating gel were transferred to polyvinylidene difluoride (PVDF) membranes at 400 mA for 40 min. After being blocked with 5% non-fat milk in Tris Buffered Saline with Tween 20 [TBST, 150 mM NaCl, 10 mM Tris (pH 7.5), and 0.05% Tween-20] at room temperature for 30 min, the blots then incubated overnight at 4 °C with the following primary antibodies: anti-HIF-1α, anti-Atg12, anti-LC3 (GeneTex, Inc., Irvine, CA), anti-SENP-1 (Abcam, Inc., Cambridge, MA, USA), anti-ARNT, anti-p62/SQSTM1, anti-beclin 1, anti-SUMO1 (Santa Cruz, Inc., Heidelberg, Germany), and anti-β-Actin (Sigma-Aldrich, St. Louis, MO, USA). Following a 30 min washing with TBST, the blots were then hybridized for 2 h at room temperature with the secondary antibody conjugated with horseradish peroxidase (HRP). Finally, protein signals were visualized with an enhanced chemiluminescence (ECL) kit (Millipore, Billerica, MA, USA) and images were captured using the BioSpectrum AC® Imaging system (UVP, Upland, CA, USA). Protein signals were quantified using the Gel-Pro analyzer software (version 4.0, Media Cybernetics, Rockville, MD, USA). Background intensity was subtracted from each sample and the sample signal was normalized to the β-actin loading control. Full paper
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