Figure 9.
Expression of the human bile acid binder (HBAB) in McNtcp.24 cells and transport of bile acids in HBAB-expressing McNtcp.24 cells (BN cells). (A) The 2.0 kb recombinant HBAB mRNA is readily detectable in total RNA (10 μg) of selected BN clones. Note the absence of the recombinant HBAB mRNA in McNtcp.24 cells. The blot was stripped and reprobed with the mouse glyceraldehyde-3-phoshate dehydrogenase (G3PDH) cDNA to demonstrate the presence of mRNA in the samples analyzed. The recombinant HBAB protein is also readily detectable by immunoblotting in total lysates (20 μg) of BN and McNtcp.24 cells. The α-actin antiserum was used to demonstrate equivalent amounts of total proteins analyzed. (B) TCA uptake activity of the BN clones and McNtcp.24 cells are shown. The values are relative to the TCA uptake activity of McNtcp.24 cells (100 %). (C) Efflux of TCA from BN clones and McNtcp.24 cells. The cells were incubated with 25 μM [3H]-TCA for 20 min to label the intracellular pool of bile acids. The amount of radioactivity that remains associated with the cells after the removal of radiolabeled TCA from the medium is shown. The values represent means ± SD (n = 3). The slopes of the regression lines were compared by analysis of variance and no statistical differences were found. The experiments in (A) and (C) were repeated twice and the experiment in (B) was repeated three times with similar results.