Nb100 122

Whole-cell BMDM protein lysate was isolated using RIPA buffer supplemented with protease inhibitors (Roche, 11873580001) and protein concentration determined by BCA assay.
Pre-cast gels (ExpressPlus PAGE gel 8–16%, genscript, M81612) were used to and proteins were transferred to a nitrocellulose membrane. Primary antibodies directed against HIF1α (Novus Biologicals, NB100-499), HIF2α (Novus Biologicals, NB100-122), and β-Actin (Abcam, ab8227), were followed by appropriate HRP-labelled secondary antibody incubation. Signal was developed using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher scientific, 34095) and visualized using a digital scanner. Band density was quantified with ImageJ, and normalized for total proteins by β-actin as loading control.

The Nuclear/Cytosol Fractionation Kit (BioVision) was used for nuclear fractionation. Extracted proteins were separated by running SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membrane. After the membrane was blocked in 5% fat-free milk in 1 × TBST and washed three times with 1 × TBST, it was immersed in 5% fat-free milk in 1 × TBST solution containing a primary antibody against HIF-2α (Novus, NB100-122; 1:200), Oct-4 (abcam, ab19857; 1 μg/ml), Nanog (ThermoFisher, PA1-097; 1:2000), Beta-tubulin (Novus, NB600-936; 1:1000), TATA binding protein (Novus, NB500-700; 1:1000), or Beta-actin (Novus, NB600-501; 1:5000), respectively at 4 °C overnight. After the membrane was washed three times with 1 × TBST, a secondary antibody against HRP-conjugated goat polyclonal anti-rabbit IgG (1:10000) or HRP-conjugated rabbit polyclonal anti-mouse IgG (1:10000) were added for 30 minutes at room temperature (RT). Finally, results were photographed by using LAS-3000 (FUJIFILM).