Transfection of AQP9 into Hep3B and K562 cells. (A) K562_EGFP-C2 cells examined under fluorescent microscopy showed even cellular distribution of green fluorescence; however, K562_EGFR-AQP9 cells showed selective localization of green fluorescence to the plasma membrane. Images were visualized using an Olympus IX70 microscope equipped with a C plan semi-apochromat 60×/0.7 numerical aperture lens (Olympus, Tokyo, Japan). A Nikon Coolpix 4500 camera (Nikon, Tokyo, Japan) was used to capture the images. (B) Western-blot analysis of Hep3B cells. In anti-GFP immunoblotting, EGFP (30 kDa) and EGFP-AQP9 (61 kDa) were detected in Hep3B_EGFP and Hep3B_EGFP-AQP9 cells, respectively. With anti-AQP9 immunoblotting, only the protein band of EGFP-AQP9 (61 kDa) was detected in Hep3B_EGFP-AQP9 cells, showing that Hep3B_EGFP cells did not express detectable levels of AQP9. (C) Western-blot analysis of K562 cells. In anti-GFP immunoblotting, protein bands of EGFP (30 kDa) and EGFP-AQP9 (61 kDa) were detected in K562_EGFP and K562_EGFP-AQP9 cells. An additional band (30-31 kDa) was observed in the K562_EGFP-AQP9 sample. This might be due to degradation of EGFP-AQP9 fusion protein into EGFP and AQP9 fractions. In anti-AQP9 immunoblotting, a predominant band of EGFP-AQP9 (61 kDa) was detected in K562_EGFP-AQP9 cells. At the anti-AQP9 antibody concentration and exposure time used to avoid overexposure of the K562E_GFP-AQP9 lysate, K562_EGFR lysate did not show detectable AQP9, as was shown previously in Figure 2.