Figure 6

Immunohistochemical evaluation of mouse Abcg5/sterolin-1 expression in liver and intestine. COS-1 cells were transiently transfected with pCMV-mouse Abcg5 or pCMV-mouse Abcg8 constructs, allowed to express for 48 hours then fixed and incubated with antibody. pCMV empty vector transfected COS-1 cells incubated with SC anti-Abcg5/sterolin-1 showed no significant fluorescence (panel a). Abcg5 transfected COS-1 cells incubated with SC anti-Abcg5/sterolin-1 antibody showed a membrane distribution (panel b), where as Abcg8 transfected cells incubated with SC anti-Abcg5 antibody showed no significant fluorescence (panel c). Abcg5 and Abcg8 co-transfected COS-1 cells incubated with SC anti-Abcg5/sterolin-1 antibody resulted in a fluorescence pattern similar to Abcg5 alone (panel d). The yellow bar represents 20 μm. Wild-type intestine incubated with SC pre-immune serum (panel e), or SC anti-Abcg5/sterolin-1 antibody pre-incubated with the blocking peptide (panel f) showed no specific signals. Wild-type, Abcg8+/- and Abcg8-/- intestine (panels g, h and i respectively) incubated with SC anti-Abcg5/sterolin-1 antibody, showed no difference in expression patterns, despite the loss of Abcg8/sterolin-2 in the knockout mice. Single arrowhead shows intestinal villus and double arrowhead shows crypt. The yellow bar represents 50 μm. Antibody staining of liver sections was also performed at the AMC Liver Center. As a control, an antibody to Bsep/Abcb11 was used and showed a clear apical distribution in both wild-type (panel j) and Abcg8 knockout mice (panel k). Using AMC antibody against Abcg5/sterolin-1 (see text), in both wild-type (panel l) and Abcg8 knockout liver (panel m), the pattern of expression was also apical and unchanged although the signal is fainter compared to that for Bsep/Abcb11 (see text for discussion). Arrows indicate bile canaliculi.