Localization of the ammonium transporters, Rh B glycoprotein and Rh C glycoprotein, in the mouse liver  I.David Weiner, R.Tyler Miller, Jill W Verlander  Gastroenterology  Volume 124, Issue 5, Pages 1432-1440 (May 2003) DOI: 10.1016/S0016-5085(03)00277-4

Figure 1 Detection of hepatic RhBG and RhCG mRNA expression using real-time RT-PCR. Mouse total RNA was reverse transcribed and then amplified using gene-specific primers and fluorescent probe. In all cases, samples were run in duplicate. Amplification of RhBG and RhCG mRNA from aliquots of the same liver sample is shown. Increases in fluorescence (Rn) with increasing amplification cycles indicate amplification of hepatic RhBG and RhCG mRNA. Greater amounts of starting mRNA yield earlier increases in Rn, indicating that expression of RhBG mRNA is greater than RhCG mRNA. Gastroenterology 2003 124, 1432-1440DOI: (10.1016/S0016-5085(03)00277-4)

Figure 2 Immunoblot of liver membrane proteins using anti-RhBG specific antibodies. Twenty micrograms of a mouse liver membrane preparation was electrophoresed on 10% polyacrylamide gel electrophoresis ReadyGel, transferred electrophoretically to nitrocellulose membranes, and blotted using anti-RhBG antibodies at 1:1000 dilution. Preincubation of the antibody with the immunizing peptide (+ peptide) prevented protein recognition in both the liver and the kidney preparation. Gastroenterology 2003 124, 1432-1440DOI: (10.1016/S0016-5085(03)00277-4)

Figure 3 Immunohistochemical localization of RhBG in the mouse liver. (A ) Low-power magnification showing that RhBG is expressed in only a subpopulation of hepatocytes. (B) High-power magnification using confocal laser scanning microscopy and showing expression of RhBG protein in a 1–2-cell-thick layer of cells surrounding a central vein (cv). Expression is limited to the plasma membrane. No expression is seen in midzonal hepatocytes or in hepatocytes surrounding a portal vein (pv). The arrow shows a hepatic artery present in the portal triad. Gastroenterology 2003 124, 1432-1440DOI: (10.1016/S0016-5085(03)00277-4)

Figure 4 Colocalization of RhBG and glutamine synthetase in the mouse liver. RhBG immunoreactivity is shown with the green label, and glutamine synthetase immunoreactivity is shown with the red label. Cells expressing plasma membrane RhBG also expressed cytosolic glutamine synthetase and vice versa. Gastroenterology 2003 124, 1432-1440DOI: (10.1016/S0016-5085(03)00277-4)

Figure 5 Immunoblot showing hepatic RhCG expression. Immunoblot of hepatic protein preparation identifies expression of low levels of RhCG protein with apparent molecular weights of ∼67 and 69 kilodaltons. No detection was observed when the antibody was preincubated with immunizing peptide. Gastroenterology 2003 124, 1432-1440DOI: (10.1016/S0016-5085(03)00277-4)

Figure 6 RhCG immunoreactivity in bile duct epithelia. Identical section of liver imaged using confocal laser scanning microscopy and differential inference contrast optics. (A ) RhCG immunoreactivity identified using a signal amplification procedure and detected using confocal laser scanning immunofluorescent microscopy. Immunoreactivity (green) was detected only in the plasma membrane of bile duct epithelia in portal triads. (B) A differential inference contrast image of the same field. Gastroenterology 2003 124, 1432-1440DOI: (10.1016/S0016-5085(03)00277-4)