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Fig. 4. Kir6.1 mRNA and protein are found in rat liver, where the Kir6.1-SUR2A complex physically interacts with components of tight junctions. (a) Immunocytochemical staining of bile canaliculi of the liver with antibodies against Kir6.1 and ZO-1. The bile canaliculi are sealed by tight junctions, which are localized between adjacent hepatocytes. Note the precise colocalization of Kir6.1 (left panel, green) and ZO-1 (middle panel, red), orange in the merged image (right panel, orange). The identical pattern of the immunoreactivities suggest the localization of Kir6.1 and SUR2A (data not shown) in bile canalicular membranes of the rat liver. Bar, 30 µm. (b) Bile-canalicular membranes were prepared from rat liver. The proteins were separated on SDS-PAGE, transferred to nitrocellulose and incubated with antisera against occludin, Kir6.1 and SUR2A. (c) Immunoprecipitations with antisera against SUR2A and Kir6.1 led to a co-precipitation of occludin, indicating that the Kir6.1-SUR2A complex physically interacts with the tight-junction-protein complex. Negative control without primary antibody (first lane); immunoprecipitation with anti-occludin used as a positive control (third lane). (d) Additional immunprecipitation with anti-occludin antiserum shows the ratio of the precipitated or co-precipitated protein (Ip) in comparison with the amount of protein still present in the supernatant (Sup). Please note that occludin and the Kir6.1 and SUR2A protein pair subunits were co-precipitated in almost equal amounts. Analysed as a negative control was AE2, which does not associate with tight junctions. As expected, AE2 is only present in the supernatant. (e) Expression of Kir6.1 mRNA in rat liver is also proven by RT-PCR.
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