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nckx4 cdna  (Addgene inc)


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    Addgene inc nckx4 cdna
    FIGURE 1. Generation of <t>Nckx4/</t> knock-out mice. A, diagram illustrating the Nckx4 (slc24a4) locus, the targeting vector, and the targeted locus. The location of Nckx4 exons 5, 6, 7, and 8 is indicated. The neomycin (neo) gene driven by a pol II promoter replaced exons 6 and 7 in the targeted locus. The thymidine kinase gene driven by a herpes simplex virus promoter was used for negative selection. The location of the probe used for Southern analysis and the length of the two diagnostic NsiI fragments of 9.9 and 4.9 kb are illustrated at the top and bottom of the diagram. The position of the primers used for PCR analysis, P1, P2, and P3, which generate diagnostic fragments of 175or350bparealsoindicated.B,genotypeanalysisbySouthernblot(left)or PCR (right) of DNA from wild-type (/), heterozygous (/), or Nckx4 knock-out(/)animals.C,Northernblot(leftpanel)analysisof10goftotal RNA samples, or immunoblot (right panel) analysis of 20 g of membrane proteins samples, isolated from the indicated tissues of mice of different genotype.
    Nckx4 Cdna, supplied by Addgene inc, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/nckx4+cdna/Human+NCKX4-AS-long+(pcDNA3%2E1%2B)+(Plasmid+%2375211)/10__1074_slash_jbc__m114__564450-76-30-18
    Average 85 stars, based on 1 article reviews
    nckx4 cdna - by Bioz Stars, 2026-09
    85/100 stars

    Images

    1) Product Images from "An Essential Role for the K+-dependent Na+/Ca2+-exchanger, NCKX4, in Melanocortin-4-receptor-dependent Satiety"

    Article Title: An Essential Role for the K+-dependent Na+/Ca2+-exchanger, NCKX4, in Melanocortin-4-receptor-dependent Satiety

    Journal: Journal of Biological Chemistry

    doi: 10.1074/jbc.m114.564450

    FIGURE 1. Generation of Nckx4/ knock-out mice. A, diagram illustrating the Nckx4 (slc24a4) locus, the targeting vector, and the targeted locus. The location of Nckx4 exons 5, 6, 7, and 8 is indicated. The neomycin (neo) gene driven by a pol II promoter replaced exons 6 and 7 in the targeted locus. The thymidine kinase gene driven by a herpes simplex virus promoter was used for negative selection. The location of the probe used for Southern analysis and the length of the two diagnostic NsiI fragments of 9.9 and 4.9 kb are illustrated at the top and bottom of the diagram. The position of the primers used for PCR analysis, P1, P2, and P3, which generate diagnostic fragments of 175or350bparealsoindicated.B,genotypeanalysisbySouthernblot(left)or PCR (right) of DNA from wild-type (/), heterozygous (/), or Nckx4 knock-out(/)animals.C,Northernblot(leftpanel)analysisof10goftotal RNA samples, or immunoblot (right panel) analysis of 20 g of membrane proteins samples, isolated from the indicated tissues of mice of different genotype.
    Figure Legend Snippet: FIGURE 1. Generation of Nckx4/ knock-out mice. A, diagram illustrating the Nckx4 (slc24a4) locus, the targeting vector, and the targeted locus. The location of Nckx4 exons 5, 6, 7, and 8 is indicated. The neomycin (neo) gene driven by a pol II promoter replaced exons 6 and 7 in the targeted locus. The thymidine kinase gene driven by a herpes simplex virus promoter was used for negative selection. The location of the probe used for Southern analysis and the length of the two diagnostic NsiI fragments of 9.9 and 4.9 kb are illustrated at the top and bottom of the diagram. The position of the primers used for PCR analysis, P1, P2, and P3, which generate diagnostic fragments of 175or350bparealsoindicated.B,genotypeanalysisbySouthernblot(left)or PCR (right) of DNA from wild-type (/), heterozygous (/), or Nckx4 knock-out(/)animals.C,Northernblot(leftpanel)analysisof10goftotal RNA samples, or immunoblot (right panel) analysis of 20 g of membrane proteins samples, isolated from the indicated tissues of mice of different genotype.

    Techniques Used: Knock-Out, Plasmid Preparation, Virus, Selection, Diagnostic Assay, Western Blot, Membrane, Isolation

    FIGURE 2. Nckx4 knock-out does not change brain morphology or the expression of other Na/Ca2-exchangers. A, serial coronal sections through the hypothalamus of wild-type adult mice were labeled for Nckx4 transcripts with an antisense or a sense control probe, as indicated. B, hematoxylin and eosin-stained coronal paraffin sections through the PVN (wild-type, /; Nckx4 knock-out, /). No differences were observed between genotypes in this or in other brain regions examined in either coronal or parasagittal stained sections (data not shown). In both panels A and B, the right-hand images are magnified views of the area in the box in the corresponding left-hand images. Scale bars: left-hand images, 1 mm; right-hand images, 200 m. C, Northern blots of 10 g of total RNA samples isolated from adult brain of wild-type (/), heterozygous (/), or Nckx4 knock-out (/) mice were analyzed with probes for the indicated Na/Ca2-exchangers genes. The blots were then stripped and reprobed for Gapdh as a loading control. All images in this figure are representative of independent experiments from 3 different animals of each genotype.
    Figure Legend Snippet: FIGURE 2. Nckx4 knock-out does not change brain morphology or the expression of other Na/Ca2-exchangers. A, serial coronal sections through the hypothalamus of wild-type adult mice were labeled for Nckx4 transcripts with an antisense or a sense control probe, as indicated. B, hematoxylin and eosin-stained coronal paraffin sections through the PVN (wild-type, /; Nckx4 knock-out, /). No differences were observed between genotypes in this or in other brain regions examined in either coronal or parasagittal stained sections (data not shown). In both panels A and B, the right-hand images are magnified views of the area in the box in the corresponding left-hand images. Scale bars: left-hand images, 1 mm; right-hand images, 200 m. C, Northern blots of 10 g of total RNA samples isolated from adult brain of wild-type (/), heterozygous (/), or Nckx4 knock-out (/) mice were analyzed with probes for the indicated Na/Ca2-exchangers genes. The blots were then stripped and reprobed for Gapdh as a loading control. All images in this figure are representative of independent experiments from 3 different animals of each genotype.

    Techniques Used: Knock-Out, Expressing, Labeling, Control, Staining, Northern Blot, Isolation

    FIGURE 3. Nckx4/ mice are anorexic and hypophagic. Knock-out (/) and wild-type (/) and heterozygous (/) littermates, as indicated, were fed ad libitum either standard or enriched laboratory chow (4.5% or 9% fat). A, animals were weighed weekly from 4 weeks of age onwards (n 12–15 for each group; however, measurements were not made for all animals for all time points, all conditions or both sexes; therefore, some data points corre- spond to a smaller n value). B, animals were weighed at birth (P0; n 10) and then at weaning (n 63, 107, and 56 for wild-type, heterozygous, and knock- out, respectively). C, weight (n 8) and length (nose to anus; n 10) were measured on adult animals. D, food (n 8 for females and n 10 for males) and water consumption (n 6) were measured in adult animals. In all cases the mean S.E. is plotted. Statistical significance of differences between knock-out and wild-type is indicated where present: *, p 0.05; **, p 0.01; ***, p 0.001.
    Figure Legend Snippet: FIGURE 3. Nckx4/ mice are anorexic and hypophagic. Knock-out (/) and wild-type (/) and heterozygous (/) littermates, as indicated, were fed ad libitum either standard or enriched laboratory chow (4.5% or 9% fat). A, animals were weighed weekly from 4 weeks of age onwards (n 12–15 for each group; however, measurements were not made for all animals for all time points, all conditions or both sexes; therefore, some data points corre- spond to a smaller n value). B, animals were weighed at birth (P0; n 10) and then at weaning (n 63, 107, and 56 for wild-type, heterozygous, and knock- out, respectively). C, weight (n 8) and length (nose to anus; n 10) were measured on adult animals. D, food (n 8 for females and n 10 for males) and water consumption (n 6) were measured in adult animals. In all cases the mean S.E. is plotted. Statistical significance of differences between knock-out and wild-type is indicated where present: *, p 0.05; **, p 0.01; ***, p 0.001.

    Techniques Used: Knock-Out

    FIGURE 4. Body temperature and tissue weight. A, basal body temperature was determined rectally in free-fed or fasted adult animals (n 4–8). B, tissues were extracted from adult (5–6 months of age) animals (n 5–7), blotted dry and weighed. Fat corresponds to total abdominal cavity adipose tissue. Average values S.E. are plotted. Statistical significance of the differences between wild-type (/; WT) and Nckx4 knock-out (/; KO) is indicated where present: *, p 0.05; **, p 0.01; ***, p 0.001. C, microscopic images of hematoxylin and eosin-labeled sections from paraffin-embedded samples of abdominal fat pad or liver are shown. Scale bar, 100 m. These images are representative of samples from at last 5 pairs of animals of each genotype.
    Figure Legend Snippet: FIGURE 4. Body temperature and tissue weight. A, basal body temperature was determined rectally in free-fed or fasted adult animals (n 4–8). B, tissues were extracted from adult (5–6 months of age) animals (n 5–7), blotted dry and weighed. Fat corresponds to total abdominal cavity adipose tissue. Average values S.E. are plotted. Statistical significance of the differences between wild-type (/; WT) and Nckx4 knock-out (/; KO) is indicated where present: *, p 0.05; **, p 0.01; ***, p 0.001. C, microscopic images of hematoxylin and eosin-labeled sections from paraffin-embedded samples of abdominal fat pad or liver are shown. Scale bar, 100 m. These images are representative of samples from at last 5 pairs of animals of each genotype.

    Techniques Used: Knock-Out, Labeling

    FIGURE 5. Health parameters, motivation and olfaction in Nckx4/ knock-out and wild-type (/) mice. A, a glucose tolerance test was performed on 5–6-month-old animals following overnight fasting. Glucose (2 mg/g body weight) was injected intraperitoneally, and blood glucose was measured just prior and at intervals thereafter. Average values S.E. are plotted (n 6). B, a Kaplan Meier survival curve is plotted for 18 wild-type (/) and 13 Nckx4 knock-out (/) animals. A log-rank (Mantel-Cox) test indicates a significant difference between genotypes, p 0.05. C, cumulative food consumption was measured in adult mice immediately following a 24-h fasting period (n 11). D, time taken to find buried urine-scented litter (left) or buried food pellet (right) was measured for adult mice (n 11–13). Statistical significance of the differences between genotypes determined by analysis of variance using the Bonferroni post-test is indicated where present: *, p 0.05; ***, p 0.001.
    Figure Legend Snippet: FIGURE 5. Health parameters, motivation and olfaction in Nckx4/ knock-out and wild-type (/) mice. A, a glucose tolerance test was performed on 5–6-month-old animals following overnight fasting. Glucose (2 mg/g body weight) was injected intraperitoneally, and blood glucose was measured just prior and at intervals thereafter. Average values S.E. are plotted (n 6). B, a Kaplan Meier survival curve is plotted for 18 wild-type (/) and 13 Nckx4 knock-out (/) animals. A log-rank (Mantel-Cox) test indicates a significant difference between genotypes, p 0.05. C, cumulative food consumption was measured in adult mice immediately following a 24-h fasting period (n 11). D, time taken to find buried urine-scented litter (left) or buried food pellet (right) was measured for adult mice (n 11–13). Statistical significance of the differences between genotypes determined by analysis of variance using the Bonferroni post-test is indicated where present: *, p 0.05; ***, p 0.001.

    Techniques Used: Knock-Out, Injection

    FIGURE 7. Neurons in the PVN are activated in Nckx4/ mice. c-Fos expression was detected by immunofluorescence in sections from hypothal- amus of adult mice. WT, wild-type; KO, Nckx4/. All animals were fasted for 48 h and subsequently treated for 30 min prior to sacrifice either with glucose injection (intraperitoneally; 2 mg/g body weight) or by perfusion with Ca2- free Krebs-Henseleit solution containing 1 mM EGTA, normal Krebs-Henseleit solution containing 100 nM insulin, or normal Krebs-Henseleit solution con- taining 1 M of the MC4R antagonist, SHU9119 (SHU). The total number of c-Fos positive nuclei contained within the PVN area were counted for each section, and the averaged data S.E. from 4 to 7 independent experiments are shown in the panel at the lower right. Statistical significance is indicated where present: compared with wild-type untreated: *, p 0.05; **, p 0.01; ***, p 0.001; compared with glucose-treated wild-type: #, p 0.05; com- pared with knock-out untreated: †††, p 0.001. Scale bar, 500 m.
    Figure Legend Snippet: FIGURE 7. Neurons in the PVN are activated in Nckx4/ mice. c-Fos expression was detected by immunofluorescence in sections from hypothal- amus of adult mice. WT, wild-type; KO, Nckx4/. All animals were fasted for 48 h and subsequently treated for 30 min prior to sacrifice either with glucose injection (intraperitoneally; 2 mg/g body weight) or by perfusion with Ca2- free Krebs-Henseleit solution containing 1 mM EGTA, normal Krebs-Henseleit solution containing 100 nM insulin, or normal Krebs-Henseleit solution con- taining 1 M of the MC4R antagonist, SHU9119 (SHU). The total number of c-Fos positive nuclei contained within the PVN area were counted for each section, and the averaged data S.E. from 4 to 7 independent experiments are shown in the panel at the lower right. Statistical significance is indicated where present: compared with wild-type untreated: *, p 0.05; **, p 0.01; ***, p 0.001; compared with glucose-treated wild-type: #, p 0.05; com- pared with knock-out untreated: †††, p 0.001. Scale bar, 500 m.

    Techniques Used: Expressing, Immunofluorescence, Injection, Knock-Out

    FIGURE 6. Blood hormones and hypothalamic mediators. A, serum sam- ples from overnight fasted female (F) or male (M) animals of wild-type (/) or Nckx4 knock-out (/) genotype (n 7–10) were tested for the indicated factors. Levels are plotted normalized to the wild-type female levels in each case. Statistical significance of differences between knock-out and wild-type for each gender is indicated where present: *, p 0.05; **, p 0.01; ***, p 0.001. The values corresponding to 100% are: insulin, 1.51 ng/ml; leptin, 55.5 ng/ml; glucose, 261 mg/dl; triglycerides, 0.49 mg/ml; T4, 5.0 g/dl; cortisol, 4.1 g/dl; MSH, 110 pmol/liter. B, localization of MSH and AgRP was deter- mined by immunofluorescence in coronal sections through the hypotha- lamic region of adult female wild-type (/) or Nckx4 knock-out (/) mice. For illustrative purposes, bilaterally symmetric image halves are compared between Nckx4/ and Nckx4/ animals for sections taken at the level of the PVN and ARC. The PVN and ARC regions are circled. Scale bar, 500 m. The right-hand most image pairs are magnified views of the ARC regions from the central image pairs, illustrating the punctate nature of staining, consis- tent with packaging of peptide hormones in vesicles destined for secretion. Scale bar, 100 m. These images are representative of three independent experiments.
    Figure Legend Snippet: FIGURE 6. Blood hormones and hypothalamic mediators. A, serum sam- ples from overnight fasted female (F) or male (M) animals of wild-type (/) or Nckx4 knock-out (/) genotype (n 7–10) were tested for the indicated factors. Levels are plotted normalized to the wild-type female levels in each case. Statistical significance of differences between knock-out and wild-type for each gender is indicated where present: *, p 0.05; **, p 0.01; ***, p 0.001. The values corresponding to 100% are: insulin, 1.51 ng/ml; leptin, 55.5 ng/ml; glucose, 261 mg/dl; triglycerides, 0.49 mg/ml; T4, 5.0 g/dl; cortisol, 4.1 g/dl; MSH, 110 pmol/liter. B, localization of MSH and AgRP was deter- mined by immunofluorescence in coronal sections through the hypotha- lamic region of adult female wild-type (/) or Nckx4 knock-out (/) mice. For illustrative purposes, bilaterally symmetric image halves are compared between Nckx4/ and Nckx4/ animals for sections taken at the level of the PVN and ARC. The PVN and ARC regions are circled. Scale bar, 500 m. The right-hand most image pairs are magnified views of the ARC regions from the central image pairs, illustrating the punctate nature of staining, consis- tent with packaging of peptide hormones in vesicles destined for secretion. Scale bar, 100 m. These images are representative of three independent experiments.

    Techniques Used: Knock-Out, Immunofluorescence, Staining

    FIGURE 9. MSH-induced Ca2 signals. A, the fura-2 340/380 fluorescent exci- tation ratios are shown for individual neurons, each plotted with a uniquely colored trace, from primary hypothalamic cultures from either wild-type (WT) or Nckx4 knock-out (KO) mice. Cells were continually perfused with Hepes- buffered saline, containing either 1 M MSH or 75 mM KCl where indicated. B, the number of cells in each microscopic field that responded with a change in fura-2 ratio, as shown in panel A, following MSH treatment is plotted. Aver- ages S.E. for 18–20 microscopic fields from six independent cultures are shown. ***, p 0.001. C, GT1–7 cells were infected with lentivirus expressing mouse NCKX4, or a deleted control construct, and subsequently assayed for Ca2 response 2 days later. The fura-2 340/380 excitation ratios for individual responding cells are shown, each plotted with a uniquely colored trace. D, the average number of responding cells per field is summarized. n 9; **, p 0.01. E, GT1–7 cells in Opti-MEM were treated with vehicle only (control) or with 10 M SKF96365 or 10 M U73122 at 37 °C for 15 min with the indicated concentration of MSH, followed by extraction and determination of cAMP levels (n 5–8).
    Figure Legend Snippet: FIGURE 9. MSH-induced Ca2 signals. A, the fura-2 340/380 fluorescent exci- tation ratios are shown for individual neurons, each plotted with a uniquely colored trace, from primary hypothalamic cultures from either wild-type (WT) or Nckx4 knock-out (KO) mice. Cells were continually perfused with Hepes- buffered saline, containing either 1 M MSH or 75 mM KCl where indicated. B, the number of cells in each microscopic field that responded with a change in fura-2 ratio, as shown in panel A, following MSH treatment is plotted. Aver- ages S.E. for 18–20 microscopic fields from six independent cultures are shown. ***, p 0.001. C, GT1–7 cells were infected with lentivirus expressing mouse NCKX4, or a deleted control construct, and subsequently assayed for Ca2 response 2 days later. The fura-2 340/380 excitation ratios for individual responding cells are shown, each plotted with a uniquely colored trace. D, the average number of responding cells per field is summarized. n 9; **, p 0.01. E, GT1–7 cells in Opti-MEM were treated with vehicle only (control) or with 10 M SKF96365 or 10 M U73122 at 37 °C for 15 min with the indicated concentration of MSH, followed by extraction and determination of cAMP levels (n 5–8).

    Techniques Used: Knock-Out, Saline, Infection, Expressing, Control, Construct, Concentration Assay, Extraction

    FIGURE 8. The Nckx4/ phenotype is MC4R-dependent. Age-matched adult mice of different genotypes were compared: WT, wild-type; N4KO, Nckx4/; M4KO, Mc4r/; DKO, Nckx4/; Mc4r/. A, body weight (n 6–9); B, food consumption (n 10); C and D, c-Fos immunofluorescence in coronal hypothalamic sections of fasted animals, as described in the legend toFig.7(n4–7).InpanelsAandB,N4KO,M4KO,andDKOareallsignificantly different from WT, and in panels A, B, and D, M4KO and DKO are significantly different from N4KO, with p 0.001 in all cases. Scale bar, 500 m.
    Figure Legend Snippet: FIGURE 8. The Nckx4/ phenotype is MC4R-dependent. Age-matched adult mice of different genotypes were compared: WT, wild-type; N4KO, Nckx4/; M4KO, Mc4r/; DKO, Nckx4/; Mc4r/. A, body weight (n 6–9); B, food consumption (n 10); C and D, c-Fos immunofluorescence in coronal hypothalamic sections of fasted animals, as described in the legend toFig.7(n4–7).InpanelsAandB,N4KO,M4KO,andDKOareallsignificantly different from WT, and in panels A, B, and D, M4KO and DKO are significantly different from N4KO, with p 0.001 in all cases. Scale bar, 500 m.

    Techniques Used: Immunofluorescence

    FIGURE 11. Control of oxytocin expression in the hypothalamic paraven- tricular nucleus. Oxytocin expression was detected by immunofluorescence in adult mice. WT, wild-type; KO, Nckx4/. Animals were fasted for 48 h and then subjected to a 30-min transcardial perfusion with Krebs-Henseleit solu- tion containing 1 M MSH, 10 M SKF96365, or 10 M U73122, as indicated. The oxytocin-positive cells were scored as being of high or low staining inten- sity, and the numbers pooled across all sections from each of five animals per treatmentgroup.Thetotalnumberofcellsscoredvariedfrom522to1288per animal, but the averaged values were not different between groups (800 cells). The averaged ratios of high/low stained cells S.E. are shown in the panel at lower right. Statistical significance is indicated where present: com- pared with wild-type untreated: **, p 0.01; ***, p 0.001. Scale bar, 100 m.
    Figure Legend Snippet: FIGURE 11. Control of oxytocin expression in the hypothalamic paraven- tricular nucleus. Oxytocin expression was detected by immunofluorescence in adult mice. WT, wild-type; KO, Nckx4/. Animals were fasted for 48 h and then subjected to a 30-min transcardial perfusion with Krebs-Henseleit solu- tion containing 1 M MSH, 10 M SKF96365, or 10 M U73122, as indicated. The oxytocin-positive cells were scored as being of high or low staining inten- sity, and the numbers pooled across all sections from each of five animals per treatmentgroup.Thetotalnumberofcellsscoredvariedfrom522to1288per animal, but the averaged values were not different between groups (800 cells). The averaged ratios of high/low stained cells S.E. are shown in the panel at lower right. Statistical significance is indicated where present: com- pared with wild-type untreated: **, p 0.01; ***, p 0.001. Scale bar, 100 m.

    Techniques Used: Control, Expressing, Immunofluorescence, Staining

    FIGURE 12. A model for MC4R-dependent Ca2 signaling and the role of NCKX4. MSH binding to the MC4R is shown to active Gq, via the exchange of GTP for GDP (upper row). It is important to note that MC4R may constitutively activate Gq even in the absence of ligand (54). So activated, Gq goes on to bind and stimulate PLC-, which subsequently cleaves phosphatidylinositol 1,4-bisphosphate (PIP2) to diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). The membrane-bound diacylglycerol then activates the Ca2-selective entry channel, TRPC6, which results in an elevation of cytosolic [Ca2] and consequent neuronal activation, which may include the initiation of action potentials (15) and the release of oxytocin (16, 41) (lower row). Normally, NCKX4 acts locally to extrude the Ca2 that enters via TRPC6, thus moderating the signaling pathway so only strong signals drive neuronal activation. In the absence of this moderating influence, even weak, constitutive, signaling via MC4R can result in sufficient Ca2 entry to initiate neuronal activation.
    Figure Legend Snippet: FIGURE 12. A model for MC4R-dependent Ca2 signaling and the role of NCKX4. MSH binding to the MC4R is shown to active Gq, via the exchange of GTP for GDP (upper row). It is important to note that MC4R may constitutively activate Gq even in the absence of ligand (54). So activated, Gq goes on to bind and stimulate PLC-, which subsequently cleaves phosphatidylinositol 1,4-bisphosphate (PIP2) to diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). The membrane-bound diacylglycerol then activates the Ca2-selective entry channel, TRPC6, which results in an elevation of cytosolic [Ca2] and consequent neuronal activation, which may include the initiation of action potentials (15) and the release of oxytocin (16, 41) (lower row). Normally, NCKX4 acts locally to extrude the Ca2 that enters via TRPC6, thus moderating the signaling pathway so only strong signals drive neuronal activation. In the absence of this moderating influence, even weak, constitutive, signaling via MC4R can result in sufficient Ca2 entry to initiate neuronal activation.

    Techniques Used: Binding Assay, Membrane, Activation Assay

    Related Articles

    Plasmid Preparation:

    Article Title: An Essential Role for the K+-dependent Na+/Ca2+-exchanger, NCKX4, in Melanocortin-4-receptor-dependent Satiety
    Article Snippet: A fragment from the pWPI vector (a kind gift of Dr. Didier Trono obtained as Addgene plasmid 12254) encompassing the central polypurine tract (cPPT), internal ribosome entry site (IRES), and enhanced green fluorescent protein (EGFP) loci was inserted at the 3 end of the Nckx4 cDNA. .. A fragment comprising the CaMKIIa promoter from the pLentiCamKII-ChETA-EYFP (a kind gift of Dr. Karl Deisseroth obtained as Addgene plasmid 26967) (28) was inserted at the 5 end of the Nckx4 cDNA. .. The entire CaMKIIa-promoterNckx4-IRES-EGFP construct was then excised as an XbaIEcoRI fragment and used to replace the corresponding CaMKIIa-promoter-ChETA-EYFP fragment from the pLenti vector.



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    nckx4 cdna  (Addgene inc)
    85
    Addgene inc nckx4 cdna
    FIGURE 1. Generation of <t>Nckx4/</t> knock-out mice. A, diagram illustrating the Nckx4 (slc24a4) locus, the targeting vector, and the targeted locus. The location of Nckx4 exons 5, 6, 7, and 8 is indicated. The neomycin (neo) gene driven by a pol II promoter replaced exons 6 and 7 in the targeted locus. The thymidine kinase gene driven by a herpes simplex virus promoter was used for negative selection. The location of the probe used for Southern analysis and the length of the two diagnostic NsiI fragments of 9.9 and 4.9 kb are illustrated at the top and bottom of the diagram. The position of the primers used for PCR analysis, P1, P2, and P3, which generate diagnostic fragments of 175or350bparealsoindicated.B,genotypeanalysisbySouthernblot(left)or PCR (right) of DNA from wild-type (/), heterozygous (/), or Nckx4 knock-out(/)animals.C,Northernblot(leftpanel)analysisof10goftotal RNA samples, or immunoblot (right panel) analysis of 20 g of membrane proteins samples, isolated from the indicated tissues of mice of different genotype.
    Nckx4 Cdna, supplied by Addgene inc, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/nckx4+cdna/Human+NCKX4-AS-long+(pcDNA3%2E1%2B)+(Plasmid+%2375211)/10__1074_slash_jbc__m114__564450-76-30-18
    Average 85 stars, based on 1 article reviews
    nckx4 cdna - by Bioz Stars, 2026-09
    85/100 stars
    		  Buy from Supplier

    Image Search Results


    FIGURE 1. Generation of Nckx4/ knock-out mice. A, diagram illustrating the Nckx4 (slc24a4) locus, the targeting vector, and the targeted locus. The location of Nckx4 exons 5, 6, 7, and 8 is indicated. The neomycin (neo) gene driven by a pol II promoter replaced exons 6 and 7 in the targeted locus. The thymidine kinase gene driven by a herpes simplex virus promoter was used for negative selection. The location of the probe used for Southern analysis and the length of the two diagnostic NsiI fragments of 9.9 and 4.9 kb are illustrated at the top and bottom of the diagram. The position of the primers used for PCR analysis, P1, P2, and P3, which generate diagnostic fragments of 175or350bparealsoindicated.B,genotypeanalysisbySouthernblot(left)or PCR (right) of DNA from wild-type (/), heterozygous (/), or Nckx4 knock-out(/)animals.C,Northernblot(leftpanel)analysisof10goftotal RNA samples, or immunoblot (right panel) analysis of 20 g of membrane proteins samples, isolated from the indicated tissues of mice of different genotype.

    Journal: Journal of Biological Chemistry

    Article Title: An Essential Role for the K+-dependent Na+/Ca2+-exchanger, NCKX4, in Melanocortin-4-receptor-dependent Satiety

    doi: 10.1074/jbc.m114.564450

    Figure Lengend Snippet: FIGURE 1. Generation of Nckx4/ knock-out mice. A, diagram illustrating the Nckx4 (slc24a4) locus, the targeting vector, and the targeted locus. The location of Nckx4 exons 5, 6, 7, and 8 is indicated. The neomycin (neo) gene driven by a pol II promoter replaced exons 6 and 7 in the targeted locus. The thymidine kinase gene driven by a herpes simplex virus promoter was used for negative selection. The location of the probe used for Southern analysis and the length of the two diagnostic NsiI fragments of 9.9 and 4.9 kb are illustrated at the top and bottom of the diagram. The position of the primers used for PCR analysis, P1, P2, and P3, which generate diagnostic fragments of 175or350bparealsoindicated.B,genotypeanalysisbySouthernblot(left)or PCR (right) of DNA from wild-type (/), heterozygous (/), or Nckx4 knock-out(/)animals.C,Northernblot(leftpanel)analysisof10goftotal RNA samples, or immunoblot (right panel) analysis of 20 g of membrane proteins samples, isolated from the indicated tissues of mice of different genotype.

    Article Snippet: A fragment comprising the CaMKIIa promoter from the pLentiCamKII-ChETA-EYFP (a kind gift of Dr. Karl Deisseroth obtained as Addgene plasmid 26967) (28) was inserted at the 5 end of the Nckx4 cDNA.

    Techniques: Knock-Out, Plasmid Preparation, Virus, Selection, Diagnostic Assay, Western Blot, Membrane, Isolation

    FIGURE 2. Nckx4 knock-out does not change brain morphology or the expression of other Na/Ca2-exchangers. A, serial coronal sections through the hypothalamus of wild-type adult mice were labeled for Nckx4 transcripts with an antisense or a sense control probe, as indicated. B, hematoxylin and eosin-stained coronal paraffin sections through the PVN (wild-type, /; Nckx4 knock-out, /). No differences were observed between genotypes in this or in other brain regions examined in either coronal or parasagittal stained sections (data not shown). In both panels A and B, the right-hand images are magnified views of the area in the box in the corresponding left-hand images. Scale bars: left-hand images, 1 mm; right-hand images, 200 m. C, Northern blots of 10 g of total RNA samples isolated from adult brain of wild-type (/), heterozygous (/), or Nckx4 knock-out (/) mice were analyzed with probes for the indicated Na/Ca2-exchangers genes. The blots were then stripped and reprobed for Gapdh as a loading control. All images in this figure are representative of independent experiments from 3 different animals of each genotype.

    Journal: Journal of Biological Chemistry

    Article Title: An Essential Role for the K+-dependent Na+/Ca2+-exchanger, NCKX4, in Melanocortin-4-receptor-dependent Satiety

    doi: 10.1074/jbc.m114.564450

    Figure Lengend Snippet: FIGURE 2. Nckx4 knock-out does not change brain morphology or the expression of other Na/Ca2-exchangers. A, serial coronal sections through the hypothalamus of wild-type adult mice were labeled for Nckx4 transcripts with an antisense or a sense control probe, as indicated. B, hematoxylin and eosin-stained coronal paraffin sections through the PVN (wild-type, /; Nckx4 knock-out, /). No differences were observed between genotypes in this or in other brain regions examined in either coronal or parasagittal stained sections (data not shown). In both panels A and B, the right-hand images are magnified views of the area in the box in the corresponding left-hand images. Scale bars: left-hand images, 1 mm; right-hand images, 200 m. C, Northern blots of 10 g of total RNA samples isolated from adult brain of wild-type (/), heterozygous (/), or Nckx4 knock-out (/) mice were analyzed with probes for the indicated Na/Ca2-exchangers genes. The blots were then stripped and reprobed for Gapdh as a loading control. All images in this figure are representative of independent experiments from 3 different animals of each genotype.

    Article Snippet: A fragment comprising the CaMKIIa promoter from the pLentiCamKII-ChETA-EYFP (a kind gift of Dr. Karl Deisseroth obtained as Addgene plasmid 26967) (28) was inserted at the 5 end of the Nckx4 cDNA.

    Techniques: Knock-Out, Expressing, Labeling, Control, Staining, Northern Blot, Isolation

    FIGURE 3. Nckx4/ mice are anorexic and hypophagic. Knock-out (/) and wild-type (/) and heterozygous (/) littermates, as indicated, were fed ad libitum either standard or enriched laboratory chow (4.5% or 9% fat). A, animals were weighed weekly from 4 weeks of age onwards (n 12–15 for each group; however, measurements were not made for all animals for all time points, all conditions or both sexes; therefore, some data points corre- spond to a smaller n value). B, animals were weighed at birth (P0; n 10) and then at weaning (n 63, 107, and 56 for wild-type, heterozygous, and knock- out, respectively). C, weight (n 8) and length (nose to anus; n 10) were measured on adult animals. D, food (n 8 for females and n 10 for males) and water consumption (n 6) were measured in adult animals. In all cases the mean S.E. is plotted. Statistical significance of differences between knock-out and wild-type is indicated where present: *, p 0.05; **, p 0.01; ***, p 0.001.

    Journal: Journal of Biological Chemistry

    Article Title: An Essential Role for the K+-dependent Na+/Ca2+-exchanger, NCKX4, in Melanocortin-4-receptor-dependent Satiety

    doi: 10.1074/jbc.m114.564450

    Figure Lengend Snippet: FIGURE 3. Nckx4/ mice are anorexic and hypophagic. Knock-out (/) and wild-type (/) and heterozygous (/) littermates, as indicated, were fed ad libitum either standard or enriched laboratory chow (4.5% or 9% fat). A, animals were weighed weekly from 4 weeks of age onwards (n 12–15 for each group; however, measurements were not made for all animals for all time points, all conditions or both sexes; therefore, some data points corre- spond to a smaller n value). B, animals were weighed at birth (P0; n 10) and then at weaning (n 63, 107, and 56 for wild-type, heterozygous, and knock- out, respectively). C, weight (n 8) and length (nose to anus; n 10) were measured on adult animals. D, food (n 8 for females and n 10 for males) and water consumption (n 6) were measured in adult animals. In all cases the mean S.E. is plotted. Statistical significance of differences between knock-out and wild-type is indicated where present: *, p 0.05; **, p 0.01; ***, p 0.001.

    Article Snippet: A fragment comprising the CaMKIIa promoter from the pLentiCamKII-ChETA-EYFP (a kind gift of Dr. Karl Deisseroth obtained as Addgene plasmid 26967) (28) was inserted at the 5 end of the Nckx4 cDNA.

    Techniques: Knock-Out

    FIGURE 4. Body temperature and tissue weight. A, basal body temperature was determined rectally in free-fed or fasted adult animals (n 4–8). B, tissues were extracted from adult (5–6 months of age) animals (n 5–7), blotted dry and weighed. Fat corresponds to total abdominal cavity adipose tissue. Average values S.E. are plotted. Statistical significance of the differences between wild-type (/; WT) and Nckx4 knock-out (/; KO) is indicated where present: *, p 0.05; **, p 0.01; ***, p 0.001. C, microscopic images of hematoxylin and eosin-labeled sections from paraffin-embedded samples of abdominal fat pad or liver are shown. Scale bar, 100 m. These images are representative of samples from at last 5 pairs of animals of each genotype.

    Journal: Journal of Biological Chemistry

    Article Title: An Essential Role for the K+-dependent Na+/Ca2+-exchanger, NCKX4, in Melanocortin-4-receptor-dependent Satiety

    doi: 10.1074/jbc.m114.564450

    Figure Lengend Snippet: FIGURE 4. Body temperature and tissue weight. A, basal body temperature was determined rectally in free-fed or fasted adult animals (n 4–8). B, tissues were extracted from adult (5–6 months of age) animals (n 5–7), blotted dry and weighed. Fat corresponds to total abdominal cavity adipose tissue. Average values S.E. are plotted. Statistical significance of the differences between wild-type (/; WT) and Nckx4 knock-out (/; KO) is indicated where present: *, p 0.05; **, p 0.01; ***, p 0.001. C, microscopic images of hematoxylin and eosin-labeled sections from paraffin-embedded samples of abdominal fat pad or liver are shown. Scale bar, 100 m. These images are representative of samples from at last 5 pairs of animals of each genotype.

    Article Snippet: A fragment comprising the CaMKIIa promoter from the pLentiCamKII-ChETA-EYFP (a kind gift of Dr. Karl Deisseroth obtained as Addgene plasmid 26967) (28) was inserted at the 5 end of the Nckx4 cDNA.

    Techniques: Knock-Out, Labeling

    FIGURE 5. Health parameters, motivation and olfaction in Nckx4/ knock-out and wild-type (/) mice. A, a glucose tolerance test was performed on 5–6-month-old animals following overnight fasting. Glucose (2 mg/g body weight) was injected intraperitoneally, and blood glucose was measured just prior and at intervals thereafter. Average values S.E. are plotted (n 6). B, a Kaplan Meier survival curve is plotted for 18 wild-type (/) and 13 Nckx4 knock-out (/) animals. A log-rank (Mantel-Cox) test indicates a significant difference between genotypes, p 0.05. C, cumulative food consumption was measured in adult mice immediately following a 24-h fasting period (n 11). D, time taken to find buried urine-scented litter (left) or buried food pellet (right) was measured for adult mice (n 11–13). Statistical significance of the differences between genotypes determined by analysis of variance using the Bonferroni post-test is indicated where present: *, p 0.05; ***, p 0.001.

    Journal: Journal of Biological Chemistry

    Article Title: An Essential Role for the K+-dependent Na+/Ca2+-exchanger, NCKX4, in Melanocortin-4-receptor-dependent Satiety

    doi: 10.1074/jbc.m114.564450

    Figure Lengend Snippet: FIGURE 5. Health parameters, motivation and olfaction in Nckx4/ knock-out and wild-type (/) mice. A, a glucose tolerance test was performed on 5–6-month-old animals following overnight fasting. Glucose (2 mg/g body weight) was injected intraperitoneally, and blood glucose was measured just prior and at intervals thereafter. Average values S.E. are plotted (n 6). B, a Kaplan Meier survival curve is plotted for 18 wild-type (/) and 13 Nckx4 knock-out (/) animals. A log-rank (Mantel-Cox) test indicates a significant difference between genotypes, p 0.05. C, cumulative food consumption was measured in adult mice immediately following a 24-h fasting period (n 11). D, time taken to find buried urine-scented litter (left) or buried food pellet (right) was measured for adult mice (n 11–13). Statistical significance of the differences between genotypes determined by analysis of variance using the Bonferroni post-test is indicated where present: *, p 0.05; ***, p 0.001.

    Article Snippet: A fragment comprising the CaMKIIa promoter from the pLentiCamKII-ChETA-EYFP (a kind gift of Dr. Karl Deisseroth obtained as Addgene plasmid 26967) (28) was inserted at the 5 end of the Nckx4 cDNA.

    Techniques: Knock-Out, Injection

    FIGURE 7. Neurons in the PVN are activated in Nckx4/ mice. c-Fos expression was detected by immunofluorescence in sections from hypothal- amus of adult mice. WT, wild-type; KO, Nckx4/. All animals were fasted for 48 h and subsequently treated for 30 min prior to sacrifice either with glucose injection (intraperitoneally; 2 mg/g body weight) or by perfusion with Ca2- free Krebs-Henseleit solution containing 1 mM EGTA, normal Krebs-Henseleit solution containing 100 nM insulin, or normal Krebs-Henseleit solution con- taining 1 M of the MC4R antagonist, SHU9119 (SHU). The total number of c-Fos positive nuclei contained within the PVN area were counted for each section, and the averaged data S.E. from 4 to 7 independent experiments are shown in the panel at the lower right. Statistical significance is indicated where present: compared with wild-type untreated: *, p 0.05; **, p 0.01; ***, p 0.001; compared with glucose-treated wild-type: #, p 0.05; com- pared with knock-out untreated: †††, p 0.001. Scale bar, 500 m.

    Journal: Journal of Biological Chemistry

    Article Title: An Essential Role for the K+-dependent Na+/Ca2+-exchanger, NCKX4, in Melanocortin-4-receptor-dependent Satiety

    doi: 10.1074/jbc.m114.564450

    Figure Lengend Snippet: FIGURE 7. Neurons in the PVN are activated in Nckx4/ mice. c-Fos expression was detected by immunofluorescence in sections from hypothal- amus of adult mice. WT, wild-type; KO, Nckx4/. All animals were fasted for 48 h and subsequently treated for 30 min prior to sacrifice either with glucose injection (intraperitoneally; 2 mg/g body weight) or by perfusion with Ca2- free Krebs-Henseleit solution containing 1 mM EGTA, normal Krebs-Henseleit solution containing 100 nM insulin, or normal Krebs-Henseleit solution con- taining 1 M of the MC4R antagonist, SHU9119 (SHU). The total number of c-Fos positive nuclei contained within the PVN area were counted for each section, and the averaged data S.E. from 4 to 7 independent experiments are shown in the panel at the lower right. Statistical significance is indicated where present: compared with wild-type untreated: *, p 0.05; **, p 0.01; ***, p 0.001; compared with glucose-treated wild-type: #, p 0.05; com- pared with knock-out untreated: †††, p 0.001. Scale bar, 500 m.

    Article Snippet: A fragment comprising the CaMKIIa promoter from the pLentiCamKII-ChETA-EYFP (a kind gift of Dr. Karl Deisseroth obtained as Addgene plasmid 26967) (28) was inserted at the 5 end of the Nckx4 cDNA.

    Techniques: Expressing, Immunofluorescence, Injection, Knock-Out

    FIGURE 6. Blood hormones and hypothalamic mediators. A, serum sam- ples from overnight fasted female (F) or male (M) animals of wild-type (/) or Nckx4 knock-out (/) genotype (n 7–10) were tested for the indicated factors. Levels are plotted normalized to the wild-type female levels in each case. Statistical significance of differences between knock-out and wild-type for each gender is indicated where present: *, p 0.05; **, p 0.01; ***, p 0.001. The values corresponding to 100% are: insulin, 1.51 ng/ml; leptin, 55.5 ng/ml; glucose, 261 mg/dl; triglycerides, 0.49 mg/ml; T4, 5.0 g/dl; cortisol, 4.1 g/dl; MSH, 110 pmol/liter. B, localization of MSH and AgRP was deter- mined by immunofluorescence in coronal sections through the hypotha- lamic region of adult female wild-type (/) or Nckx4 knock-out (/) mice. For illustrative purposes, bilaterally symmetric image halves are compared between Nckx4/ and Nckx4/ animals for sections taken at the level of the PVN and ARC. The PVN and ARC regions are circled. Scale bar, 500 m. The right-hand most image pairs are magnified views of the ARC regions from the central image pairs, illustrating the punctate nature of staining, consis- tent with packaging of peptide hormones in vesicles destined for secretion. Scale bar, 100 m. These images are representative of three independent experiments.

    Journal: Journal of Biological Chemistry

    Article Title: An Essential Role for the K+-dependent Na+/Ca2+-exchanger, NCKX4, in Melanocortin-4-receptor-dependent Satiety

    doi: 10.1074/jbc.m114.564450

    Figure Lengend Snippet: FIGURE 6. Blood hormones and hypothalamic mediators. A, serum sam- ples from overnight fasted female (F) or male (M) animals of wild-type (/) or Nckx4 knock-out (/) genotype (n 7–10) were tested for the indicated factors. Levels are plotted normalized to the wild-type female levels in each case. Statistical significance of differences between knock-out and wild-type for each gender is indicated where present: *, p 0.05; **, p 0.01; ***, p 0.001. The values corresponding to 100% are: insulin, 1.51 ng/ml; leptin, 55.5 ng/ml; glucose, 261 mg/dl; triglycerides, 0.49 mg/ml; T4, 5.0 g/dl; cortisol, 4.1 g/dl; MSH, 110 pmol/liter. B, localization of MSH and AgRP was deter- mined by immunofluorescence in coronal sections through the hypotha- lamic region of adult female wild-type (/) or Nckx4 knock-out (/) mice. For illustrative purposes, bilaterally symmetric image halves are compared between Nckx4/ and Nckx4/ animals for sections taken at the level of the PVN and ARC. The PVN and ARC regions are circled. Scale bar, 500 m. The right-hand most image pairs are magnified views of the ARC regions from the central image pairs, illustrating the punctate nature of staining, consis- tent with packaging of peptide hormones in vesicles destined for secretion. Scale bar, 100 m. These images are representative of three independent experiments.

    Article Snippet: A fragment comprising the CaMKIIa promoter from the pLentiCamKII-ChETA-EYFP (a kind gift of Dr. Karl Deisseroth obtained as Addgene plasmid 26967) (28) was inserted at the 5 end of the Nckx4 cDNA.

    Techniques: Knock-Out, Immunofluorescence, Staining

    FIGURE 9. MSH-induced Ca2 signals. A, the fura-2 340/380 fluorescent exci- tation ratios are shown for individual neurons, each plotted with a uniquely colored trace, from primary hypothalamic cultures from either wild-type (WT) or Nckx4 knock-out (KO) mice. Cells were continually perfused with Hepes- buffered saline, containing either 1 M MSH or 75 mM KCl where indicated. B, the number of cells in each microscopic field that responded with a change in fura-2 ratio, as shown in panel A, following MSH treatment is plotted. Aver- ages S.E. for 18–20 microscopic fields from six independent cultures are shown. ***, p 0.001. C, GT1–7 cells were infected with lentivirus expressing mouse NCKX4, or a deleted control construct, and subsequently assayed for Ca2 response 2 days later. The fura-2 340/380 excitation ratios for individual responding cells are shown, each plotted with a uniquely colored trace. D, the average number of responding cells per field is summarized. n 9; **, p 0.01. E, GT1–7 cells in Opti-MEM were treated with vehicle only (control) or with 10 M SKF96365 or 10 M U73122 at 37 °C for 15 min with the indicated concentration of MSH, followed by extraction and determination of cAMP levels (n 5–8).

    Journal: Journal of Biological Chemistry

    Article Title: An Essential Role for the K+-dependent Na+/Ca2+-exchanger, NCKX4, in Melanocortin-4-receptor-dependent Satiety

    doi: 10.1074/jbc.m114.564450

    Figure Lengend Snippet: FIGURE 9. MSH-induced Ca2 signals. A, the fura-2 340/380 fluorescent exci- tation ratios are shown for individual neurons, each plotted with a uniquely colored trace, from primary hypothalamic cultures from either wild-type (WT) or Nckx4 knock-out (KO) mice. Cells were continually perfused with Hepes- buffered saline, containing either 1 M MSH or 75 mM KCl where indicated. B, the number of cells in each microscopic field that responded with a change in fura-2 ratio, as shown in panel A, following MSH treatment is plotted. Aver- ages S.E. for 18–20 microscopic fields from six independent cultures are shown. ***, p 0.001. C, GT1–7 cells were infected with lentivirus expressing mouse NCKX4, or a deleted control construct, and subsequently assayed for Ca2 response 2 days later. The fura-2 340/380 excitation ratios for individual responding cells are shown, each plotted with a uniquely colored trace. D, the average number of responding cells per field is summarized. n 9; **, p 0.01. E, GT1–7 cells in Opti-MEM were treated with vehicle only (control) or with 10 M SKF96365 or 10 M U73122 at 37 °C for 15 min with the indicated concentration of MSH, followed by extraction and determination of cAMP levels (n 5–8).

    Article Snippet: A fragment comprising the CaMKIIa promoter from the pLentiCamKII-ChETA-EYFP (a kind gift of Dr. Karl Deisseroth obtained as Addgene plasmid 26967) (28) was inserted at the 5 end of the Nckx4 cDNA.

    Techniques: Knock-Out, Saline, Infection, Expressing, Control, Construct, Concentration Assay, Extraction

    FIGURE 8. The Nckx4/ phenotype is MC4R-dependent. Age-matched adult mice of different genotypes were compared: WT, wild-type; N4KO, Nckx4/; M4KO, Mc4r/; DKO, Nckx4/; Mc4r/. A, body weight (n 6–9); B, food consumption (n 10); C and D, c-Fos immunofluorescence in coronal hypothalamic sections of fasted animals, as described in the legend toFig.7(n4–7).InpanelsAandB,N4KO,M4KO,andDKOareallsignificantly different from WT, and in panels A, B, and D, M4KO and DKO are significantly different from N4KO, with p 0.001 in all cases. Scale bar, 500 m.

    Journal: Journal of Biological Chemistry

    Article Title: An Essential Role for the K+-dependent Na+/Ca2+-exchanger, NCKX4, in Melanocortin-4-receptor-dependent Satiety

    doi: 10.1074/jbc.m114.564450

    Figure Lengend Snippet: FIGURE 8. The Nckx4/ phenotype is MC4R-dependent. Age-matched adult mice of different genotypes were compared: WT, wild-type; N4KO, Nckx4/; M4KO, Mc4r/; DKO, Nckx4/; Mc4r/. A, body weight (n 6–9); B, food consumption (n 10); C and D, c-Fos immunofluorescence in coronal hypothalamic sections of fasted animals, as described in the legend toFig.7(n4–7).InpanelsAandB,N4KO,M4KO,andDKOareallsignificantly different from WT, and in panels A, B, and D, M4KO and DKO are significantly different from N4KO, with p 0.001 in all cases. Scale bar, 500 m.

    Article Snippet: A fragment comprising the CaMKIIa promoter from the pLentiCamKII-ChETA-EYFP (a kind gift of Dr. Karl Deisseroth obtained as Addgene plasmid 26967) (28) was inserted at the 5 end of the Nckx4 cDNA.

    Techniques: Immunofluorescence

    FIGURE 11. Control of oxytocin expression in the hypothalamic paraven- tricular nucleus. Oxytocin expression was detected by immunofluorescence in adult mice. WT, wild-type; KO, Nckx4/. Animals were fasted for 48 h and then subjected to a 30-min transcardial perfusion with Krebs-Henseleit solu- tion containing 1 M MSH, 10 M SKF96365, or 10 M U73122, as indicated. The oxytocin-positive cells were scored as being of high or low staining inten- sity, and the numbers pooled across all sections from each of five animals per treatmentgroup.Thetotalnumberofcellsscoredvariedfrom522to1288per animal, but the averaged values were not different between groups (800 cells). The averaged ratios of high/low stained cells S.E. are shown in the panel at lower right. Statistical significance is indicated where present: com- pared with wild-type untreated: **, p 0.01; ***, p 0.001. Scale bar, 100 m.

    Journal: Journal of Biological Chemistry

    Article Title: An Essential Role for the K+-dependent Na+/Ca2+-exchanger, NCKX4, in Melanocortin-4-receptor-dependent Satiety

    doi: 10.1074/jbc.m114.564450

    Figure Lengend Snippet: FIGURE 11. Control of oxytocin expression in the hypothalamic paraven- tricular nucleus. Oxytocin expression was detected by immunofluorescence in adult mice. WT, wild-type; KO, Nckx4/. Animals were fasted for 48 h and then subjected to a 30-min transcardial perfusion with Krebs-Henseleit solu- tion containing 1 M MSH, 10 M SKF96365, or 10 M U73122, as indicated. The oxytocin-positive cells were scored as being of high or low staining inten- sity, and the numbers pooled across all sections from each of five animals per treatmentgroup.Thetotalnumberofcellsscoredvariedfrom522to1288per animal, but the averaged values were not different between groups (800 cells). The averaged ratios of high/low stained cells S.E. are shown in the panel at lower right. Statistical significance is indicated where present: com- pared with wild-type untreated: **, p 0.01; ***, p 0.001. Scale bar, 100 m.

    Article Snippet: A fragment comprising the CaMKIIa promoter from the pLentiCamKII-ChETA-EYFP (a kind gift of Dr. Karl Deisseroth obtained as Addgene plasmid 26967) (28) was inserted at the 5 end of the Nckx4 cDNA.

    Techniques: Control, Expressing, Immunofluorescence, Staining

    FIGURE 12. A model for MC4R-dependent Ca2 signaling and the role of NCKX4. MSH binding to the MC4R is shown to active Gq, via the exchange of GTP for GDP (upper row). It is important to note that MC4R may constitutively activate Gq even in the absence of ligand (54). So activated, Gq goes on to bind and stimulate PLC-, which subsequently cleaves phosphatidylinositol 1,4-bisphosphate (PIP2) to diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). The membrane-bound diacylglycerol then activates the Ca2-selective entry channel, TRPC6, which results in an elevation of cytosolic [Ca2] and consequent neuronal activation, which may include the initiation of action potentials (15) and the release of oxytocin (16, 41) (lower row). Normally, NCKX4 acts locally to extrude the Ca2 that enters via TRPC6, thus moderating the signaling pathway so only strong signals drive neuronal activation. In the absence of this moderating influence, even weak, constitutive, signaling via MC4R can result in sufficient Ca2 entry to initiate neuronal activation.

    Journal: Journal of Biological Chemistry

    Article Title: An Essential Role for the K+-dependent Na+/Ca2+-exchanger, NCKX4, in Melanocortin-4-receptor-dependent Satiety

    doi: 10.1074/jbc.m114.564450

    Figure Lengend Snippet: FIGURE 12. A model for MC4R-dependent Ca2 signaling and the role of NCKX4. MSH binding to the MC4R is shown to active Gq, via the exchange of GTP for GDP (upper row). It is important to note that MC4R may constitutively activate Gq even in the absence of ligand (54). So activated, Gq goes on to bind and stimulate PLC-, which subsequently cleaves phosphatidylinositol 1,4-bisphosphate (PIP2) to diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). The membrane-bound diacylglycerol then activates the Ca2-selective entry channel, TRPC6, which results in an elevation of cytosolic [Ca2] and consequent neuronal activation, which may include the initiation of action potentials (15) and the release of oxytocin (16, 41) (lower row). Normally, NCKX4 acts locally to extrude the Ca2 that enters via TRPC6, thus moderating the signaling pathway so only strong signals drive neuronal activation. In the absence of this moderating influence, even weak, constitutive, signaling via MC4R can result in sufficient Ca2 entry to initiate neuronal activation.

    Article Snippet: A fragment comprising the CaMKIIa promoter from the pLentiCamKII-ChETA-EYFP (a kind gift of Dr. Karl Deisseroth obtained as Addgene plasmid 26967) (28) was inserted at the 5 end of the Nckx4 cDNA.

    Techniques: Binding Assay, Membrane, Activation Assay