Figure 2.

BSEP distributed with endosomal markers in subcellular fractions. WIF-B9 cells infected with BSEP-YFP adenovirus were labeled with sNHS-LC biotin at 4°C, washed, homogenized, and postnuclear supernatants were separated on sucrose step gradients. Aliquots of each fraction were analyzed by SDS-PAGE and Western blotting with antibodies against BSEP, rab4, 5, and 11, and EEA-1 or streptavidin. Fraction 1, homogenization buffer/8% sucrose interphase; fraction 2, 8%/12% interphase; fraction 3, 12%/16% interphase; fraction 4, 16%/19% interphase; fraction 5, 19%/23% interphase; fraction 6, 23%/27% interphase; fraction 7, 27%/31% interphase; fraction 8, 35%/43% interphase; and fraction 9, pelleted material. Bands on gels were quantified by densitometry, and results are expressed as percentage of total amount of each marker in the gradient.