4.3. Protein Extraction and Western Blot Analysis

Whole protein was extracted from MDA-MB-231 cells. Cells were grown in T25 flasks; when confluency reached 80%, cells were exposed to a hypoxia chamber of 1% O₂ and/or treated with 0.5 mM DMOG for the indicated time. The cell lysate was done quickly to save HIF-1α from degradation by adding 200 μL of RIPA buffer mixed with protease inhibitor (100:1) on the ice plate. Following that, a PCA kit (Merck, Darmstadt, Germany) was used to determine the concentration of the protein. Each sample contained 20 μg of total protein, which was then loaded and separated by 10% SDS-PAGE and blotted on a PVDF membrane. To block the membranes, 5% of BSA with tris buffered saline–Tween 20 (TBS-T) was added for one hour in a shaker, and then incubated overnight at 4 °C on the shaker with the following primary antibodies and dilutions: HIF-1α (1:1000, BD Biosciences, Franklin Lakes, New Jersey, US), MMP-2, VEGF, Cool/β-PIX (1:1000, Cell Signaling Technology, Danvers, MA, USA), and β-actin (1:10000, Santa Cruz, CA, USA); antibodies were diluted with TBS-T buffer containing 5% BSA. Following that, the primary antibody was discarded and washed 3 times with TBS-T each time for 10 min. Membranes were then incubated with HRP-conjugated secondary antibodies for one hour (1:10000 for β-actin and 1:1000 for other antibodies). Then, PVDF membranes were washed with TBS-T three times, each time for 10 min, and viewed under chemiluminescence using a gel-documentation system (Vilber Lourmat, Vilber, Marne-la-Vallée, France) after incubating membranes with enhanced chemiluminescent reagents (ECL). Band quantification was measured using ImageJ software.