Ab84593

Tissues were homogenized in pre-cooled RIPA Lysis Buffer (P0013B, Beyotime, China) and subjected to extraction of total proteins using centrifugation. Protein concentrations were determined by the BCA method. For all samples, the equal amount of proteins was loaded and separated using SDS-PAGE (8% gel). After gel electrophoresis, proteins were electrophoretically transferred to a polyvinylidene difluoride (PVDF) filter membrane (0.45 mm, Millipore, USA). To prevent nonspecific staining, the membrane was treated with a blocking buffer containing 5% non-fat dry milk in TBST buffer. The target proteins were blotted with primary antibodies against HIF-1α, GAPDH (ab84593, ab65979; and ab9485; diluted 1:1000 by Tris Buffer Saline or TBS; Abcom, Hong Kong), and p-Akt (sc-7976; diluted 1:500 by TBS; Santa Cruz Biotechnology, USA) and incubated overnight at 4°C. The following day blotted proteins were incubated with IgG horseradish peroxidase-linked secondary antibodies (diluted 1:1000 by TBS). Protein blotted in the membrane was visualized after being infiltrated with enhanced chemiluminescence plus reagent (ECL kit, Ewbio, China).