Western blots hepatocytes

Figure 6 Correlation between testosterone 6P-hydroxylation and CYP3A protein levels, as determined by Western blot, in human hepatocytes incubated with several prototypical inducers.

Figure 15 CYP3A potency indices of DFP in cultured rat hepatocytes as determined by Western blots, testosterone 6(3-hydroxylation, and DFP turnover. Abbreviation DFP, [(5,5-dimethyl-3-(2-propoxy)-4-(4-methanesulfonylphenyl)-2(5//)-furanone)J. Source From Ref. 64.

P-gp is constitutively expressed in nearly all barrier tissues. Techniques involving Northern blots (37) or Western blots with monoclonal antibodies such as C219 (38) and MRK 16 (39) have been used extensively to determine the tissue distribution of P-gp. It is expressed in adrenal cortex, kidney, liver, intestine, and pancreas endothelial cells at blood-tissue barriers, namely, the CNS, the testis, and in the papillary dermis (3,4,38,40,41). P-gp displays specific subcellular localization in cells with a polarized excretion or absorption function. More specifically, P-gp is found at the apical (AP) canalicular surface of hepatocytes, in the AP membrane of the columnar epithelial cells of colon and jejunum, and the AP brush border of the renal proximal tubule epithelium (3,4,40 1-2). In endothelial cells, P-gp is located in the luminal membrane (4,43). 

Figure 1. Effect of mild trypsin digestion on CPT I activity in permcaldized hepatocytes. Hepatocytes were preincubaled for 15 min in the absence (open circles) or in the presence (filled ciicles) of O.SpM OA. ( Ib were then permeabilized with digitonin and thorougiy washed with digitonin-free medium as described in ref. Per meabilized hepatocytes were subsequently resuspended at 1.5-2.0 mg piotdii mT and treated with varying con centrations of trypsin at 4 C. Trypsin action was stopped after 2 min and CPT I activity was subsequently determined in those permeabilized hepatocytes. Results correspond to 4 different cell preparations. Inset Mitochondria were isolated from permeabilized hepatocytes that had been treated without (lane a) or with (lane b) 17.5 pgmT trypsin and CPT I was detected by Western blotting. The arrow points to the 88 kDa band.

In order to ascertain that the loss in the poly(ADP-ribose) transferase band was due to a reduction in the amount of enzyme present in the hepatocytes of treated animals, rat liver extracts were analyzed by the western blot technique (9) using a polyclonal antibody against poly(ADP-ribose) transferase (donated by C. Niedergang). The protein band of 116... 

Figure 1. Expression of CYP lAl in P NF-treated rat hepatocytes. Hepatocytes were isolated from P-naphthoflavone (f-NF) and com oil (ControlJ-treated rats. After culture for 2 or 20 h, microsomal proteins were prepared from hepatocytes and loaded onto a 10% SDS-polyacrylamide gel (10 pg protein, each). (A) CYP lA level by Western blotting (B) EROD activity were measured as described in Materials and methods. Values are presented as means S.D.

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