Expression and functional analysis of Jk proteins inXenopus oocytes.

(A) Time course of [¹⁴C]-urea uptake inXenopus oocytes injected with 0.1 ng cRNA encoding the wt Jk and Jk(S291P) polypeptides. As negative control, water-injected oocytes were used. The assay was initiated by suspending individual oocytes in 0.2 mL Barth solution containing 8 μCi/mL [¹⁴C]-urea (145 μmol/L) and 5 μCi/mL [³H]-raffinose. The urea uptake was stopped after the indicated time (t = 0, 1.5, 3, and 6 minutes) by addition of 3 mL of ice-cold Barth solution and followed by rapid washes with 2 × 5 mL of the same solution. Then, the oocyte-associated [¹⁴C]-radioactivity was determined as previously described.6 Six to 10 individual oocytes were counted for each time. Mean and SE from different experiments are shown. (B) Northern blot analysis of cRNA encoding wt Jk and Jk(S291P) in oocytes. Total oocyte RNAs (15 μg/lane) at the day of injection and 3 days after were separated and hybridized with the [³²P]-labeled Jk cDNA probe, as described in “Materials and methods.” Equal loading and absence of degradation were checked by staining with ethidium bromide. Autoradiography was for 1 hour at −80°C. (C) Immunostaining of oocyte sections. Oocytes from the same batches were fixed and sections of injected oocytes were stained with an affinity-purified antibody against the N-terminus of the Kidd/urea transporter protein and visualized with FITC-conjugated anti-rabbit IgG as described in Materials and methods and then imaged using Nikon Eclipse TE300 microscope (Nikon, Paris, France). Images were taken with epifluorescence illumination (magnification, × 40) and treated with a Biocom informatic system of image integration.