Organ-specific alterations in RARα:RXRα abundance regulate rat Mrp2 (Abcc2) expression in obstructive cholestasis  Lee A. Denson, Alan Bohan, Matthew A. Held, James L. Boyer  Gastroenterology  Volume 123, Issue 2, Pages 599-607 (August 2002) DOI: 10.1053/gast.2002.34758 Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 1 Mrp2 expression is suppressed in liver and preserved in kidney in obstructive cholestasis. (A) RPA was performed by using total RNA from 1-, 3-, and 14-day sham and BDL liver and kidney and probes for rat Mrp2 and 28S. (C) Immunoblot was performed by using membrane proteins from 1-day sham and BDL liver and a polyclonal Mrp2 antibody. Representative blots at each time point are shown. (B, D) Mrp2 signal intensity relative to 28S for the RPA, or Mrp2 alone for the immunoblot, was determined by densitometry and is shown. N = 3 at each time point for sham and BDL, *P < 0.01 by ANOVA, for BDL value vs. sham control. (B)...■..., BDL kidney;—■—, BDL liver. (D) OD, optical density. Gastroenterology 2002 123, 599-607DOI: (10.1053/gast.2002.34758) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 2 RARα:RXRα binding to the Mrp2 promoter is reduced after BDL. (A) EMSA was performed by using nuclear protein from 1-, 3-, and 14-day sham and BDL liver and 14-day sham and BDL kidney and a probe for the rat Mrp2 promoter RARα:RXRα cis element. Representative blots at each time point are shown. (B) Signal intensity was determined by densitometry and normalized to sham values at each time point. N = 3 at each time point for sham and BDL, *P < 0.01 by ANOVA for BDL value vs. sham control. (C) EMSA competition and supershift assays were also performed as shown. COMP, oligonucleotide competitor; SP, specific 100-fold excess of RARE unlabeled oligonucleotide; NSP, nonspecific 100-fold excess of unrelated Sp1/Sp3 consensus unlabeled oligonucleotide, L2A; AB, antibody used for supershift assay.20 Gastroenterology 2002 123, 599-607DOI: (10.1053/gast.2002.34758) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 3 RARα and RXRα nuclear protein abundance is reduced after BDL in liver and preserved in kidney. Immunoblots were performed by using nuclear protein from (A) 1-, 3-, and 14-day sham and BDL liver and (C) kidney and polyclonal antibodies for RXRα, RARα, and SH-PTP1. Representative blots at each time point are shown. (B) Signal intensity was determined by densitometry and normalized to sham values at each time point. N = 3 at each time point for sham and BDL, *P < 0.01 by ANOVA for BDL value vs. sham control. OD, optical density normalized to sham value. (B) Solid line, RXRα; dotted line, RARα. Gastroenterology 2002 123, 599-607DOI: (10.1053/gast.2002.34758) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 4 RARα and RXRα RNA expression is reduced after BDL in liver. RPA was performed by using total RNA from 1-, 3-, and 14-day sham and BDL liver and probes for RXRα, RARα, and 28S. (A) Representative blots at each time point are shown. (B) Signal intensity was determined by densitometry and normalized to sham values at each time point. N = 3 at each time point for sham and BDL, *P < 0.01 by ANOVA for BDL values vs. sham control. (B)...■..., RARα;—■—, RXRα. Gastroenterology 2002 123, 599-607DOI: (10.1053/gast.2002.34758) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 5 Mrp2 RNA expression is reduced by IL-1β in primary rat hepatocytes. (A) Enzyme-linked immunosorbent assay for rat IL-1β was performed by using tissue homogenates from 1-day sham and BDL liver and kidney. (B) RPA for Mrp2 was performed by using total RNA isolated from primary rat hepatocytes 24 hours after treatment with IL-1β (100 ng/mL) or CDCA (50 μmol/L). Representative blots are shown. (C) Signal intensity was determined by densitometry and is shown. N = 4–5 for each treatment condition, *P < 0.01 by ANOVA for IL-1β abundance in BDL liver vs. sham liver and for Mrp2 RNA level in IL-1β–treated hepatocytes vs. untreated hepatocytes. PG/MG, picogram IL-1β/mg tissue; OD, optical density. Gastroenterology 2002 123, 599-607DOI: (10.1053/gast.2002.34758) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 6 RXRα nuclear protein abundance is reduced by IL-1β in primary rat hepatocytes. (A) Immunoblots were performed by using nuclear protein isolated from primary rat hepatocytes 24 hours after treatment with IL-1β (100 ng/mL) or CDCA (50 μmol/L) and polyclonal antibodies for RXRα, RARα, and SH-PTP1. Representative blots are shown. (B) Signal intensity was determined by densitometry and is shown. N = 4–5 for each treatment condition, *P < 0.01 by ANOVA for RXRα abundance in IL-1β–treated hepatocytes vs. untreated hepatocytes. OD, optical density; C, control. Gastroenterology 2002 123, 599-607DOI: (10.1053/gast.2002.34758) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 7 RXRα and RARα RNA expression is reduced by IL-1β in primary rat hepatocytes. RPA was performed by using total RNA isolated from primary rat hepatocytes 24 hours after treatment with IL-1β (100 ng/mL) or CDCA (50 μmol/L) and probes for RXRα, RARα, and 28S. (A) Representative blots are shown. (B) Signal intensity was determined by densitometry. N = 4–5 for each treatment condition, *P < 0.01 by ANOVA for RARα or RXRα RNA level in IL-1β–treated hepatocytes vs. untreated hepatocytes, and for RXRα RNA level in CDCA-treated hepatocytes vs. untreated hepatocytes. OD, optical density. Gastroenterology 2002 123, 599-607DOI: (10.1053/gast.2002.34758) Copyright © 2002 American Gastroenterological Association Terms and Conditions