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fig1.tif2224KHepatic CYP2E1, CYP1A2, CYP3A11 (rat CYP3A2) levels. Immunoblot of hepatic CYP2E1, CYP1A2, and CYP3A11 in LBP wildtype (C57Bl/6) vs. LBP knockout (LBP KO) mice 24 hours after intraperitoneal saline (Control) or acetaminophen (350 mg/kg) (Acetaminophen).
fig2a.tif2166KAdenoviral-mediated expression of LBP in vitro. The recombinant LBP adenovirus was constructed with the assistance of the University of Michigan Gene Vector Core and tested for activity and expression in vitro. Briefly, using polymerase chain reaction the secretory tag sequence from the plasminogen activator inhibitor type 1 gene was inserted one amino acid upstream of the histidine tag from the baculovirus transfer vector, pBlueBacHis2 (Invitrogen, Carlsbad, CA). The PCR product containing both the secretory tag and the histidine tag was subcloned into the adenoviral shuttle vector pACCMVplmpl-SSP that contains a CMV promoter. The Xho/HindIII segment of the previously constructed rat LBP cDNA in pBlue BacHis28 containing the translation start site and the entire coding sequence was subcloned into the adenoviral shuttle vector. Recombinant adenovirus using the Ad5 strain with a dl309 backbone was then produced by University of Michigan Gene Vector Core. For the control, the same adenovirus backbone containing an irrelevant protein, b-galactosidase, was used In vitro expression of LBP with recombinant adenovirus containing LBP. (A) Immunoblot of the cell pellets from 293 cells infected with 12 adenoviral-LBP clones using anti-Xpress antibody (Invitrogen). Samples were prepared with freeze thaw lysis.
fig2b.doc23KAdenoviral-mediated expression of LBP in vitro (cont.). (B) LBP bioactivity was examined using a assay which measured reactivity of RAW267.4 cells to LPS. TNF-a production in response to LPS by RAW 267.4 monocytic cells in the presence of either media (Media), supernatant (Ad-LBP Sup) or lysates of the cell pellets (Ad-LBP pellet) from adenoviral clone 10 infected 293 cells. 3% supernatant from baculovirus produced recombinant LBP (rLBP) was used as a positive control. Freeze thaw lysis was carried out on the Ad-LBP pellets to solubilize the pellet. *P < .01 by ANOVA between media and Ad-LBP sup, Ad-LBP pellet, or rLBP.
fig3a.tif624KAdenoviral expression of LBP in vivo. (A) Immunoblot for recombinant LBP 6 and 24 hours after acetaminophen in LBP knockout mice given either adenovirus containing the control protein (B) or LBP (L). Plasma from 3 mice in each group (Ad-Bgal or Ad-LBP) was electrophoresced. Membranes were probed with horse peroxidase labeled anti-His antibodies (Invitrogen).
fig3b.doc23KAdenoviral expression of LBP in vivo (cont.). (B) Plasma levels of rat LBP measured by ELISA 6 and 24 hours after acetaminophen in LBP knockout mice given either adenovirus containing the control protein (Ad-Bgal) or LBP (Ad-LBP). No detectable rat LBP was noted in the mice given Ad-Bgal.

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