On day 1, MEF cells (+/+, -/-), were grown on 15cm dish (2x10 to the 6th cells). On day 2, cells were exposed to hypoxia for 4hrs. Cells were washed with ice cold PBS twice and whole cell protein was extracted with RIPA buffer fortified with protease. Upon quantification, 100 µg of protein was fractionated on 7% polyacralymide gel. -Gel was transferred overnight onto nitrocellulose membrane. The membrane was probed with HIF-1 alpha monoclonal antibody at a 1:500 dilution. The secondary antibody was conjugated with HRP and was used at a 1:2500 dilution.