Volume 120, Issue 2, Pages 525-533 (February 2001) Organic anion-transporting polypeptide B (OATP-B) and its functional comparison with three other OATPs of human liver  Gerd A. Kullak-Ublick, Manfred G. Ismair, Bruno Stieger, Lukas Landmann, Robert Huber, Flavia Pizzagalli, Karin Fattinger, Peter J. Meier, Bruno Hagenbuch  Gastroenterology  Volume 120, Issue 2, Pages 525-533 (February 2001) DOI: 10.1053/gast.2001.21176 Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 1 Tissue distribution of OATP-B. Multiple-tissue Northern blots containing 2 μg poly(A)+ RNA per lane were hybridized with an OATP-B cDNA probe and, after high-stringency washing (see Materials and Methods), were exposed to autoradiography film at −70°C for 12 hours. Gastroenterology 2001 120, 525-533DOI: (10.1053/gast.2001.21176) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 2 (A) Molecular mass determination and (B) immunolocalization of OATP-B. (A) A total of 100 μg of human liver microsomal protein was separated by 7.5% SDS–polyacrylamide gel electrophoresis and transferred to nitrocellulose. Blots were incubated with either OATP-B antiserum (lane 1) or preimmune antiserum (lane 2). Bound antibodies were visualized with 125I-labeled protein A. (B) Semithin cryosections of human liver were used for indirect immunofluorescence (see Materials and Methods) and probed with antiserum against OATP-B (green) and monoclonal antibody (C219) recognizing canalicular antigens (red). Labeling was visualized with secondary antibodies conjugated to Cy2 and TxR, respectively. OATP-B was expressed exclusively at the basolateral membrane of hepatocytes, because there was no overlap of OATP-B with canalicular antigens (arrows). Intracellular globular structures were detected by both channels as a yellow signal and correspond to lysosomal residual material (lipofuscin). (C) Control sections that were not exposed to antiserum confirmed that this signal is caused by nonspecific autofluorescence. Bar = 25 μm. Gastroenterology 2001 120, 525-533DOI: (10.1053/gast.2001.21176) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 2 (A) Molecular mass determination and (B) immunolocalization of OATP-B. (A) A total of 100 μg of human liver microsomal protein was separated by 7.5% SDS–polyacrylamide gel electrophoresis and transferred to nitrocellulose. Blots were incubated with either OATP-B antiserum (lane 1) or preimmune antiserum (lane 2). Bound antibodies were visualized with 125I-labeled protein A. (B) Semithin cryosections of human liver were used for indirect immunofluorescence (see Materials and Methods) and probed with antiserum against OATP-B (green) and monoclonal antibody (C219) recognizing canalicular antigens (red). Labeling was visualized with secondary antibodies conjugated to Cy2 and TxR, respectively. OATP-B was expressed exclusively at the basolateral membrane of hepatocytes, because there was no overlap of OATP-B with canalicular antigens (arrows). Intracellular globular structures were detected by both channels as a yellow signal and correspond to lysosomal residual material (lipofuscin). (C) Control sections that were not exposed to antiserum confirmed that this signal is caused by nonspecific autofluorescence. Bar = 25 μm. Gastroenterology 2001 120, 525-533DOI: (10.1053/gast.2001.21176) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 3 BSP uptake in OATP-B, OATP-C, and OATP8 cRNA-injected oocytes. Oocytes were injected with 5 ng cRNA (●) or water (○). After 3 days in culture, initial (15-minute) uptake of [35S]BSP was measured at 25°C at the indicated concentrations. Uptake values represent means ± SEM of 8–12 individual oocyte measurements. OATP-specific uptake (dashed line) was calculated after subtracting nonspecific uptake by water-injected oocytes. Data were fitted and kinetic parameters calculated by nonlinear regression analysis. Gastroenterology 2001 120, 525-533DOI: (10.1053/gast.2001.21176) Copyright © 2001 American Gastroenterological Association Terms and Conditions