XB-IMG-126181
Xenbase Image ID: 126181
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Figure 2. Expression, localization and function of recombinant AQP1 and KCC4 in X. laevis oocytes.(A) Cell surface biotinylation experiments and subsequent Western blot analyses using anti-HA antibodies indicated plasma membrane expression of multi-AQP1 and multi-KCC4 (i.e. AQP1 and KCC4 containing a long N-terminal extension as described in Figure 1A): see multi-AQP1 monomer band below the 37 kDa marker, and multi-KCC4 monomer and dimer bands below the 150 kDa and above the 250 kDa markers, respectively. (B) Confocal immunofluorescence microscopy using anti-HA antibodies localized multi-AQP1 and multi-KCC4 in the plasma membrane. These representative images were recorded at 10x magnification. Scale bar: 200 µm. (C) Functional characterization of different AQP1 constructs. Swelling in water of non-injected oocytes (Control) and oocytes injected with cRNA of wt-AQP1 (no N-terminal extension), HA-AQP1 (only N-terminal HA epitope) and multi-AQP1. Multi-AQP1 was not functional while wt-AQP1 and HA-AQP1 had comparable activities. Data correspond to oocyte volume variation measurements in water at different time points. They are shown as averages (± S.E.) of 4-6 experiments. (D) Functional characterization of different KCC4 constructs. Similar levels of Rb+ influx into wt-KCC4 and multi-KCC4 expressing oocytes indicated full activity of the recombinant transporter. Data correspond to Rb+ influxes and are shown as averages (± S.E.) of 4 experiments (9-15 oocytes/experiment). Oocytes were non-injected (Controls), or injected with 20 ng of AQP1 and KCC4 cRNAs for expression and localization studies (A and B), and injected with 5 ng of AQP1 and 20 ng of KCC4 cRNAs for functional studies (C and D). Image published in: Bergeron MJ et al. (2011) Bergeron et al. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license Larger Image Printer Friendly View |