Rapid HIF-1α Stabilization in Whole Cell Lysates

HIF-1α protein is rapidly degraded by the ubiquitin-proteasome system under normoxic conditions^{49 (link)}. In order to avoid further HIF-1α degradation during whole cell lysates preparation, cells were lysed directly with NuPAGE® LDS Sample Buffer (1X) (Thermo Fisher Scientific, San Jose, CA, USA) in the presence of protease and phosphatase inhibitors cocktail (Cell Signaling Technology, Danvers, MA, USA), similarly to ref. 50 (link). Protein extracts were separated by electrophoresis on 4–12% NuPAGE Bis-Tris Mini Gel and then transferred onto nitrocellulose membranes using iBlot transfer stacks (Life Technologies, Gaithersburg, MD, USA). Membranes were probed with various primary antibodies overnight at 4 °C. All washes and secondary antibody incubations were performed using the SNAP i.d. quick immunoblot vacuum system (Millipore, Billerica, MA, USA). Bands were developed using Clarity Western ECL substrate (Bio-Rad Laboratories, Hercules, CA, USA). Visualization and quantitative densitometry analysis were performed with Molecular Imager® Gel Doc™ XR System v5.2.1 (Bio-Rad Laboratories, Hercules, CA, USA).