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FIGURE 1. Structural features of fNKCC2. A, regulatory region in the N terminus of NKCC1 and NKCC2. Ferret (f) sequences are aligned with their human (hu) counterparts. Known phosphorylation sites are marked by shading. Peptides used to raise phosphospecific antibodies R5 (30) and anti-pNKCC (13) are shown by boxes. B, amino acid sequences of fNKCC2 variants are encoded by alternative splicing of the 96-bp cassettes of exon 4. fNKCC2 sequences are aligned with huNKCC2, with differences between the isoforms indicated by shading. The second transmembrane domain is underlined.
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FIGURE 2. Interaction of fNKCC2 variants with antibodies. A and B, whole cell lysates (7 μg) of untransfected (−) HEK-293 cells and cells transfected with fNKCC2A, -B, -F, and -AF; and C, crude membranes (adjusted to allow similar levels of detection) from ferret (FK) and rat (RK) kidneys, were subjected to SDS-PAGE and immunoblotted with antibodies (see Table 1 for details) against NKCC1 (N1), NKCC2 (anti-NKCC2, L224, and N2-Ct), NKCC1 and 2 (T4), and phoshpo-NKCC1 and 2 (R5). Molecular mass markers (in kDa) are shown on the left and NKCC isoforms on the right. A, monomeric NKCC. B, high molecular weight bands. The molecular weight calibration in the top and bottom panels are identical. C, comparison of NKCCs in kidney and fNKCC2A-transfected HEK-293 cells.
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FIGURE 3. fNKCC2 glycosylation and appearance in the cell surface. A, whole cell lysates from HEK-293 cells transfected with fNKCC2A, -B, and -F were treated with (+) or without (−) PNGase F, subjected to SDS-PAGE, and immunoblotted with anti-NKCC2 (left panel) and re-probed with T4 (right panel). Exposure time was increased to detect fNKCC2F (shown by vertical line). B, detection of surface proteins. 48 h after transfection HEK-293 cells were surface biotinylated with Sulfo-NHS-LC-biotin. Biotinylated proteins were precipitated from cell lysates with streptavidin beads, eluted, run on SDS-PAGE, and immunoblotted with anti-NKCC2 (left panels) and re-probed with N1 to detect endogenous NKCC1 (right panels). Twice as much protein was loaded and longer exposure times were used to detect surface proteins compared with total proteins. Molecular mass markers (in kDa) are indicated.
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FIGURE 4. Functional expression of fNKCC2 variants in HEK-293 cells. HEK-293 cells were transfected with empty vector (pcDNA) or fNKCC2 constructs (A, B, and F) and grown to confluence. 2–3 days post-transfection, 86Rb uptake was assessed following preincubation in isotonic (basal conditions, open bars) or hypotonic/low-chloride medium (filled bars). In each experiment, 86Rb uptakes were normalized to the 86Rb uptake in untransfected HEK-293 cells under basal conditions and shown as mean ± S.E. Uptake (in nanomole mg protein−1 min−1) under basal conditions were 4.0 ± 0.4 (n = 19) and in specific experiments were: 4.7 ± 0.8 (versus pcDNA), 4.2 ± 0.6 (versus fNKCC2A), 4.1 ± 0.5 (versus fNKCC2B), and 3.6 ± 0.5 (versus fNKCC2F). Transfection of HEK-293 cells increased bumetanide-sensitive 86Rb uptake by 80 ± 17% (-A, n = 9), 144 ± 26% (-B; n = 10), and 53 ± 13% (-F; n = 10) under basal conditions and by 39 ± 10% (-A, n = 5), 82 ± 6% (-B, n = 5), and 50 ± 6% (-F, n = 5) following incubation in a hypotonic/low-chloride medium. *, p < 0.05; **, p < 0.005 compared with HEK-293 uptake under the same condition. Uptakes in the presence of 10 μm bumetanide were similar under all conditions. Thus between 14 and 35% total uptake was bumetanide-insensitive depending on whether the cells had been incubated in hypotonic/low chloride or isotonic media. Expression and phosphorylation of NKCC proteins were assessed by immunoblot analysis using the antibodies indicated.
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FIGURE 5. Stable expression of fNKCC2. A, HEK-293 cells were transfected with pCI-fNKCC2 constructs (A, B, and F) or empty vector (−) and selected for geneticin resistance. Equal amounts of whole cell lysates were subjected to SDS-PAGE, immunoblotted with anti-NKCC2 (top panel), and re-probed with N1 (bottom panel). Molecular size markers (in kDa) are indicated. B, HEK-293 cells either untransfected, or stably expressing fNKCC2A, were grown to confluence, and 86Rb uptake was assessed following incubation of cells in isotonic (basal conditions, open bars) or hypotonic/low-chloride (filled bars) medium. Bumetanide-sensitive 86Rb uptakes are shown as mean ± S.E. Stable expression of fNKCC2A increased 86Rb uptake by 145 ± 30% (n = 17, p = 0.00028) in basal conditions and by 72 ± 10% (n = 16, p = 0.00013) following incubation in hypotonic/low-chloride medium. Expression and phosphorylation of NKCC proteins were assessed using the antibodies indicated.
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FIGURE 6. fNKCC2AF: glycosylation, surface expression, and 86Rb uptake. A, cell lysates from HEK-293 cells transfected with fNKCC2AF were incubated with (+) or without (−) PNGase F, subjected to SDS-PAGE, immunoblotted with anti-NKCC2 (left panel), and re-probed with N1 antibody (right panel). Molecular size markers (in kDa) and cross-reaction of anti-NKCC2 with NKCC1 (*) are indicated on the left and NKCC2AF is marked by an arrow on the right. B, 48 h after transfection with fNKCC2AF HEK-293 cells were surface biotinylated with Sulfo-NHS-LC-biotin. Biotinylated proteins were precipitated from cell lysates with streptavidin beads, run on SDS-PAGE, immunoblotted with anti-NKCC2 (left), and re-probed with N1 to detect endogenous NKCC1 (right). Twice as much protein was loaded and longer exposure times were used to detect surface proteins (S) compared with total proteins (T). C, untransfected (HEK) and fNKCC2AF-transfected (AF) HEK-293 cells, and D, stable fNKCC2A (A) and stable fNKCC2A cells co-expressing fNKCC2AF (A+AF) were grown to confluence (2–3 days). 86Rb uptake was assessed following incubation of cells in isotonic (basal conditions, open bars) or hypotonic/low-chloride (filled bars) medium. Bumetanide-sensitive 86Rb uptakes are shown as mean ± S.E. Expression of fNKCC2AF has no significant (n.s.) effect on 86Rb uptakes by HEK-293 cells (C) (n ≥ 4) or HEK-293 cells stably expressing fNKCC2A (D) (n ≥ 3).
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FIGURE 7. Comparison of NKCC1 and fNKCC2 by two-dimensional gel electrophoresis. Whole cell lysates from untransfected, fNKCC2AF-transfected HEK-293 cells, and cells stably expressing fNKCC2A were separated on 7-cm Immobiline DryStrips pH 3–10, the strips were subjected to SDS-PAGE followed by immunoblotting with anti-NKCC2 and re-probing with N1 antibody. Representative blots are shown. The molecular masses of complexes exceeded 300 kDa. The pH gradient across the strip, calculated according to information provided by the manufacturer, is given at the top.
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