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Figure 2. Sequence alignment of novel NBCn2 from different species.(A) Alignment of the 3â²-portion of exon 2 of mouse Slc4a10 and homologous exons of SLC4A10 from other species. The two vertical gray bars indicate the two potential start codons for translation. An âAâ to âGâ transition mutation occurs in the first âATGâ codon in mouse Slc4a10. Note that shown here is the sequence derived from mouse strain C57BL/6J. An analysis on the genomic sequence (accession# AAHY01016664.1) showed that this same mutation is also present in Slc4a10 from mouse strain 129X1/SvJ. Also note that, compared to the non-rodent species, a âTGTâ insertion (underlined) occurs in mouse and rat following the first start codon. (B) Alignment of the predicted amino-acid sequences of the novel Nt of NBCn2 encoded by exon 2 of SLC4A10 from different species. The alignments of DNA and amino-acid sequences were performed with multiple sequence alignment tool ClustalW2 from the European Bioinformatics Institute (http://www.ebi.ac.uk/Tools/msa/clustalw2/) followed by a manual adjustment. The asterisks â*â indicate positions of fully conserved residue. The colons â:â indicate conservation between groups of strongly similar properties, whereas the periods â.â indicate conservation between groups of weakly similar properties. (C) Phylogenetic tree based upon the DNA sequence alignment shown in panel A.
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Figure 3. Diagram of primary structures of NBCn2 variants.The unique portion (16 aa) of the MEIK-NBCn2 Nt are represented in blue. The unique Nt of novel rat and mouse NBCn2 variants are represented in green. Compared to mouse, the rat novel Nt contains an additional extension of 13 aa residues (see Figure 2B). Insert A (30 aa, orange) is encoded by exon 9 in Figure 1A. The unique long Cts (21 aa, indicated by yellow) of NBCn2-C, -D, -G, and -H contain a PDZ-binding motif âETCLâ. The unique sequences of the two non-PDZ-Ct are indicated at the right end. Ala256 (position 256 relative to the first Met of MEIK-NBCn2) may be omitted in MEIK-NBCn2 [25] as well as the novel NBCn2 variants identified in the present study (i.e., rat NBCn2-F and rat NBCn2-H). The diagram was drawn to scale (scale bar: 100 aa). The sequence alignment was based on human NBCn2-A (accession# NP_071341), human NBCn2-B (#AAQ83632), rat NBCn2-C (#AAO59639), mouse NBCn2-D (#ADX99207), rat NBCn2-E (#AFP48940), rat NBCn2-F (#AFP48941), rat NBCn2-G (#AFP48942), rat NBCn2-H (#AFP48943), mouse NBCn2-I (#AFQ60533), mouse NBCn2-J(#AFN27376).
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Figure 4. Variations and alternative splicing of exon 27 (insert B).Originally described to contain 39 nt [15], the new insert B contains 60 nt and can be spliced by four different mechanisms, leading to the expression of four different NBCn2 Ct. The diagram shows the exon structures encoding the four types of NBCn2 Ct. The unique Ct amino-acid sequences are shown at the right end of each transcript. Insert B contains two stop codons (indicated by stars). Splicing-in of the 17-nt insert B leads to expression of the short âRSâ Ct using the second stop codon. Splicing-in of either the 39-nt or the 60-nt insert B leads to the expressionâusing the first stop codonâof two NBCn2 Ct, both ending with an âSSPSâ motif but differing in the presence/absence of a 7-aa cassette âRLHSFAIâ. Finally, skipping of the entire exon extends the open reading frame to exon 28, resulting in the expression of the longest NBCn2 Ct containing a PDZ-binding motif âETCLâ. The numbers in the boxes indicate the length of insert B.
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Figure 5. Functional characterization of NBCn2 variants.The oocytes were superfused with nominally HCO3â-free ND96. An acid load was introduced by exposing the cells to 1.5% CO2/10 mM HCO3â which was followed by a removal of extracellular Na+. Intracellular pH and membrane potential Vm of the oocytes were simultaneously recorded with microelectrodes. Representative recordings of pHi and Vm are shown for rNBCn2-C-EGFP (A), rNBCn2-G-EGFP (B), rNBCn2-GÎNt-EGFP (C), and H2O-injected control oocyte (D). (E) Summary of pHi recovery rates (dpHi/dt) of oocytes expressing NBCn2 variants/mutant or injected with H2O. The dpHi/dt of NBCn2-expressing oocytes are all significantly different from that of H2O-injected control oocytes.
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Figure 6. Characterization of mouse Slc4a10 promoters.(A) Diagram of the minimal promoter regions of mouse Slc4a10. Slc4a10 contains two alternative promoters: the distal P1 and the proximal P2. Shown here are only the first three exons (E1âE3) of Slc4a10. The gray areas indicate the coding regions of the exons. The numbers indicate the nucleotide positions relative to the start codon âATGâ, with ââ1â representing the first nucleotide upstream of the start codon encoding âMEIKâ or âMQPGâ of mouse NBCn2. The arrows indicate the transcription initiation site. The connections between the exons indicate the splicing of Slc4a10 transcripts produced from the alternative promoters. Exon 2 is omitted in the mature Slc4a10 transcripts produced from P1. (B) Characterization of the distal promoter (P1) expressing mMEIK-NBCn2. (C) Characterization of the proximal promoter (P2) expressing mMQPG-NBCn2. For transcription activity assay, the genomic sequence was subcloned into pGL3 vector containing firefly luciferase reporter gene. The constructs with âRâ indicate that the corresponding sequences were subcloned into pGL3 in reverse direction. A plasmid containing the renilla luciferase gene was simultaneously transfected with pGL3 containing the firefly luciferase gene. The ratios of the fluorescence intensity of firefly to that of renilla were used as indices of the transcription activities. Each bar represents the mean of at least three independent experiments. Quadruplicates were prepared for each construct in each experiment.
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Figure 7. Quantitative PCR analysis of the relative abundances of transcripts encoding mMEIK-NBCn2 and mMQPG-NBCn2 in mouse brain.(A) Representative amplification plot of qPCR for the transcripts encoding the total of mMEIK-NBCn2 in mouse brain. (B) Representative amplification plot of qPCR for the transcripts encoding the total of mMQPG-NBCn2 in mouse brain. (C) Summary of threshold cycles (CT) for mMEIK-NBCn2 and mMQPG-NBCn2. The amplification efficiencies were 91.7±2.4% (nâ=â5) for mMEIK-NBCn2, and 93.1±3.5% (nâ=â5) for mMQPG-NBCn2, pâ=â0.76 by student's t-test. The data in panel C represent the summarized CT for the undiluted cDNA samples obtained from four experiments like those shown in panels A and B. ÎRn: normalized reporter signal of qPCR products subtracted by the background signal. Two-tailed student's t-test was performed for statistical comparison between the amplification efficiencies and CT values of mMEIK- vs. mMQPG-NBCn2.
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Figure 8. Expression of transcripts encoding novel NBCn2 variants in mouse tissues.(A) Diagram to show the first four exons of the transcripts encoding the novel NBCn2 variants. Exon numbers (consistent with the numbering in Figure 1A) are marked on the top. The open area of the bar indicates the non-coding region of exon 2, whereas the gray areas represent the coding regions. The arrows indicate the approximate locations of the two sets of primers used for the nested RT-PCR. (B) RT-PCR analysis of transcripts encoding the novel NBCn2 variants in mouse tissues. By nested RT-PCR, a product of â¼340 bp was obtained from the brain, heart, kidney, male as well as female reproductive tract (RT) tissues. The male RT represented the mixture of testis and epididymis. The female RT represented the mixture of ovary, uterus, and vagina. H2O was used as template for control.
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Figure 9. Validation of polyclonal antibodies, expression and deglycosylation of NBCn2 in kidney.(A) Validation of NBCn2 antibodies by western blotting with GST-fusion proteins. The whole lysate of bacteria expressing the GST-fusion proteins were separated by two parallel 10% SDS-PAGE, and transferred onto two PVDF membranes. (B) Validation of anti-MCDL by western blotting with the protein preparations from rat kidney. The lane âTotalâ represents 300 µg of whole lysate, whereas the âMembraneâ and âSolubleâ represent the pellet and supernatant obtained from an amount of whole lysate equivalent to that loaded in lane âTotalâ. (C) Deglycosylation of NBCn2 proteins expressed in rat kidney. The protein preparations were treated in the presence or absence of Glycopeptidase F (GPF). In the experiments for panels B and C, the proteins were separated on 8% SDS-PAGE. All antibodies were used at a dilution of 1â¶5000.
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Figure 10. Expression of NBCn2 proteins in rat tissues.(A) Expression of NBCn2 with the originally described Nt in rat tissues. (B&C) Expression of NBCn2 with the novel Nt in rat tissues. For each tissue, equal amount (50 µg) of membrane proteins were separated on 8% SDS-PAGE, and blotted onto PVDF membrane. Two parallel blots were simultaneously prepared, and probed with anti-MEIK or anti-MCDL at a dilution of 1â¶5000.
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Figure 11. Tissue-specificity of expression of transcripts encoding mouse NBCn2 variants with alternative Ct.(A) Exon structures of the Ct portion of NBCn2 variants. The transcripts encoding NBCn2 with non-PDZ-Ct contain insert B (exon 27), whereas those encoding NBCn2 with PDZ-Ct lack insert B. The exon numbers (consistent with the numbering in Figure 1A) are indicated on the top of each box. The gray bars represent the coding regions, whereas the open boxes represent the UTRs. The arrows indicate the primers used in the RT-PCR analysis. (B) RT-PCR analysis of the expression of insert B in brain tissues. (C) RT-PCR analysis of the expression of insert B in the kidney, male as well as female reproductive tract (RT). The female reproductive tract included the mixture of the ovary, uterus, and vagina. The male reproductive tract included the mixture of the testis, epididymis, and vas deferens. The PCR products obtained with a plasmid containing mouse NBCn2-A (with insert B) or NBCn2-C (lacking insert B) were served as MW markers.
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Figure 1. Cloning of novel NBCn2 variants.(A) Structure of the updated mouse Slc4a10 gene. The updated mouse Slc4a10 contains 28 exons (represented by vertical bars), among which the 2nd was newly identified in the present study. The white areas of the bars represent the UTRs. The exon numbers are indicated on the top of the bars. The two triangles indicate the two major cassette exons—exon 9 (insert A) and exon 27 (insert B)—that can be alternatively spliced in or out. Arrows a and b indicate the approximate positions of the anti-sense primers used for the 5′-RACE. The diagram was drawn to scale (scale bar: 10 kb). (B) Amplification of 5′-UTR of Slc4a10 transcripts by nested 5′-RACE from mouse brain. The 1st lane represents the product of unnested RACE reaction, whereas the 2nd lane represents the nested PCR products. (C) Cloning of the full-length cDNA encoding NBCn2 variants containing the novel Nt from mouse brain tissues. (D) Cloning of the full-length cDNA encoding NBCn2 variants containing the novel Nt from the whole rat brain tissues. Nested RT-PCR was performed to amplify the full-length cDNA. H2O was used as the template for control.
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