Fig. 6.

Western blot and immunofluorescent detection of hemoglobin. A) Cytosolic and nuclear extracts were prepared from normal NCM460 and cancerous (SW620) colon cells which had been exposed to 150 μM hemoglobin for the times shown. The samples were subjected to SDS-PAGE followed by immunoblotting. The membranes were probed using antibodies against hemoglobin-α (red, 14kDa), GAPDH (green, 37 kDa) and simultaneous two-color fluorescent secondary antibodies and analyzed on an Odyssey-infrared imaging system (LI-COR®). Both cytosolic and nuclear cell extracts expressed GAPDH at similar levels. The cytosolic protein extracts expressed Hb-α at similar levels while the nuclear extracts expressed hemoglobin-α in a more time-dependent manner. B) Immunofluorescent staining of normal cells incubated for 4 h in complete media without or with 100 μM Hb. The cells were treated with Prolong® Gold antifade reagent with DAPI for visualization of the nucleus using a blue filter while the autofluorescent properties of Hb were visualized using a red filter. Both frames show nuclear and cytosolic images superimposed at a 30 × magnification; Hb uptake into both the cytosol and nucleus is shown.