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HEP_23089_sm_SupFig1.tif3731KSupplementary Figure 1. Generation of bsep−/−/mdr1a−/−/b−/− (TKO) mice: A. Crossing scheme. The mdr1a−/−/b−/− double knockout and bsep−/− mice were used to generate triply heterozygotic bsep+/−/ mdr1a+/−/b+/− mice (100% of offspring are triple heterozygotes). The triple heterozygotes were used to produce bsep+/−/mdr1a−/−/b−/− mice (approximately 1/8 of the offspring since the mdr1a and mdr1b genes in mice are closely linked), which were then used to generate the TKO homozygotes (bsep−/−/mdr1a−/−/b−/−). B. A PCR screening result for the TKO mice. #1, #5 and #6 are triple knockout mice, from which only bands from mutant alleles were amplified.
HEP_23089_sm_SupFig2.tif3731KSupplementary Figure 2. A. The major alternatively spliced Mdr1b transcript in the TKO mice. This transcript has an exon 4 deletion and results in translation of only 38 original Mdr1b amino acids followed by a frame shift, 6 novel amino acids and a premature stop codon. B. The minor Mdr1b transcript in TKO mice. This transcript has a deletion of exons 4, 5, and 6 that results in translation of 38 original amino acids followed by a frame shift, 12 novel amino acids and a premature stop codon. C. Western blotting of the adrenal glands from mdr1a−/−/b−/−, bsep−/−/mdr1a−/−/b−/− (TKO) and wild-type (WT) mice, showing the null expression of Mdr1b in TKO mice by loss of C219 antibody staining.
HEP_23089_sm_SupFig3.tif15401KSupplementary Figure 3. Liver hemorrhage seen in a dying TKO male at the age of 50 days. A dark red blood clot can be seen in the liver.
HEP_23089_sm_SupFig4.tif4058KSupplementary Figure 4. Western blotting of the apoptosis-related proteins Caspase-3, Bax, Bcl-2 and phosphor-Bad (Ser112) in the livers of triple knockout (bsep−/−/mdr1a−/−/b−/− TKO) mice in comparison with bsep−/−, mdr1a−/−/b−/− and wild-type mice. Equal amounts of liver lysate from individual animals were resolved by SDS-PAGE and analyzed by Western blotting using specific antibodies. The blots were re-probed with anti-β -actin antibody to confirm protein loading. Primary antibodies (anti-Caspase 3 Cat# 9662, anti-Bcl 2 Cat#2876, anti-Bax Cat#2772 and anti-Phospho-Bad (Ser112) Cat#9291) were all from Cell Signaling Technology, Inc. (Beverly, MA).

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