Hif 1α antibody

ChIP was performed as previously described (Polo et al., 2008 (link)). Briefly, ChIPs for HIF-1α were performed with 10 to 20 × 10⁶ Caki-2 cells under normoxic and hypoxic conditions using 5 ug HIF-1α antibody (Novus, NB100-134). An IgG antibody (Upstate Biotechnology) was used as a negative control. Enriched DNA fragments were detected by qPCR and are presented as enrichment relative to input.

For confocal imaging, 5 X 10⁵ Mϕ were plated on Millicell 4-well glass EZ slides (Millipore) and treated as described above. Lysosomal imaging was performed 24h post IL-4 (10 ng/ml) stimulation. Mϕ were exposed to 25 nM Lysotracker Red DND-99 (Life Technologies) for 30 min prior to imaging. Cells were washed twice with Hanks Balanced Salt Solution (HBSS) containing 1% FBS and imaged immediately.
For MT3 analysis, Mϕ were stimulated with IL-4 for 24h, next day, media was changed and cells were restimulated with IL-4 for 9h, followed by fixation, permeabilization and staining with MT3 antibody (Novus Biologicals). For HIF1α analysis, Mϕ were treated with siRNA for 24h, followed by IL-4 stimulation for 24h. Where indicated, cells were treated with 1 mM L-lactate or 10 mM DCA on both days of culture period. Cells were probed with HIF1α antibody (Novus Biologicals) and Alexa Fluor 647 was used as the secondary antibody (Life Technologies). Cells were mounted using VectaShield with DAPI (Vector Labs) and images were acquired with a 63X oil immersion/1.4 NA objective and 1 – 1.5 mm optical thickness on a Zeiss LSM710 confocal connected to Zeiss Axio-observer.Z1 inverted microscope and visualized using ZEN 2011 software. MFI of MT3 and nuclear localization signal of HIF1α were analyzed using ImageJ software.