Western blot using Hif3a polyclonal antibody (Cat # PAB10271) shows detection of aband ~72 KDa corresponding to mouse Hif3a (arrowhead). Approximately 10 ug of a CoCl2 treated NIH/3T3 cell lysate (Lane 1) and control NIH/3T3 cell lysate (Lane 2) were separated by 4-20% SDS-PAGE and transferred onto nitrocellulose. Treatment of exponentially growing NIH/3T3 cells with 130 uM CoCl2 for 6 h at 37°C effectively mimicshypoxia. After blocking the membrane was probed overnight at 4°C with the primary antibody diluted to 1:1,600. The membrane was washed and reacted with a 1:10,000 dilution of IRDye™800 conjugated Gt-a-Rabbit IgG [H&L] MX for 45 min at room temperature. IRDye™800 fluorescence image was captured using the Odyssey® Infrared Imaging System developed by LI-COR. IRDye is a trademark of LI-COR, Inc.