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Addgene inc human nfatc1 cdna

A- The missense SNPs lead to a P66L substitution at the N-terminal region of the protein near the calcineurin-docking site (Cln binding), and to a I701L substitution at the C-terminal region downstream of the Rel Homolgy Domain (RHD). The schematic represents isoform A, the most abundant <t>NFATC1</t> protein with 717 amino acids, a transactivation domain (TAD) at the N-terminus, and a DNA binding domain at the C-terminus. (NLS = nuclear localization signal, NES = nuclear export signal, and SP = Serine-Proline). B- The NFATC1 secondary structures were predicted and visualized by the Discovery Studio program (Acclerys Inc.). The results demonstrate the formation of a new beta-sheet in the P66L mutant and a deletion of a beta-sheet in the I701L mutant as compared to the wild type protein.

Human Nfatc1 Cdna, supplied by Addgene inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/cdna+for+nfatc1/sugarless+cDNA+(Plasmid+%2342063)/pmc03511479-59-22-25
Average 90 stars, based on 1 article reviews

human nfatc1 cdna - by Bioz Stars, 2026-09

90/100 stars

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1) Product Images from "Two Heterozygous Mutations in NFATC1 in a Patient with Tricuspid Atresia"

Article Title: Two Heterozygous Mutations in NFATC1 in a Patient with Tricuspid Atresia

Journal: PLoS ONE

doi: 10.1371/journal.pone.0049532

Figure Legend Snippet: A- The missense SNPs lead to a P66L substitution at the N-terminal region of the protein near the calcineurin-docking site (Cln binding), and to a I701L substitution at the C-terminal region downstream of the Rel Homolgy Domain (RHD). The schematic represents isoform A, the most abundant NFATC1 protein with 717 amino acids, a transactivation domain (TAD) at the N-terminus, and a DNA binding domain at the C-terminus. (NLS = nuclear localization signal, NES = nuclear export signal, and SP = Serine-Proline). B- The NFATC1 secondary structures were predicted and visualized by the Discovery Studio program (Acclerys Inc.). The results demonstrate the formation of a new beta-sheet in the P66L mutant and a deletion of a beta-sheet in the I701L mutant as compared to the wild type protein.

Techniques Used: Binding Assay, Mutagenesis

Figure Legend Snippet: Frequency of the NFATC1 mutations according to the Exome Sequencing Project (ESP).

Techniques Used: Sequencing

A- Immunofluorescence of HeLa cells transfected with plasmids encoding for the Wt NFATC1 and NFATC1 Mutants (P66L, I701L, P66L/I701L). The localization of NFATC1 was visualized using an anti-Flag antibody. Nuclei of cells were visualized using the Hoechst dye (blue color). Wt and NFATC1 mutants localized to the cytoplasm in the absence of PPP3CA (red color). (Magnification ×40). B- Immunofluorescence of HeLa cells transfected with plasmids encoding for the Wt NFATC1 and NFATC1 Mutants (P66L, I701L, P66L/I701L) co-transfected with PP3CA. The localization of NFATC1 was visualized using an anti-Flag antibody (red color) while PP3CA was visualized using anti-HA antibody (green color). Nuclei of cells were visualized using Hoechst dye (blue color). Most of the cells co-transfected with the double NFATC1 mutant were retained in the cytoplasm around the nuclear membrane, whereas in the other cases, the protein was totally translocated to the nucleus. (Magnification ×40). Yellow arrows indicate cytoplasmic (peri-nuclear) staining, while red arrows indicate nuclear staining.

Figure Legend Snippet: A- Immunofluorescence of HeLa cells transfected with plasmids encoding for the Wt NFATC1 and NFATC1 Mutants (P66L, I701L, P66L/I701L). The localization of NFATC1 was visualized using an anti-Flag antibody. Nuclei of cells were visualized using the Hoechst dye (blue color). Wt and NFATC1 mutants localized to the cytoplasm in the absence of PPP3CA (red color). (Magnification ×40). B- Immunofluorescence of HeLa cells transfected with plasmids encoding for the Wt NFATC1 and NFATC1 Mutants (P66L, I701L, P66L/I701L) co-transfected with PP3CA. The localization of NFATC1 was visualized using an anti-Flag antibody (red color) while PP3CA was visualized using anti-HA antibody (green color). Nuclei of cells were visualized using Hoechst dye (blue color). Most of the cells co-transfected with the double NFATC1 mutant were retained in the cytoplasm around the nuclear membrane, whereas in the other cases, the protein was totally translocated to the nucleus. (Magnification ×40). Yellow arrows indicate cytoplasmic (peri-nuclear) staining, while red arrows indicate nuclear staining.

Techniques Used: Immunofluorescence, Transfection, Mutagenesis, Staining

A- NFATC1 extracts from HEK 293 cells transfected with Wt NFATC1 and Mutants (P66L – I701L – P66L/I701L) were resolved on an SDS-PAGE prior to gel shift assays. Western blots showed equal amounts of expressed proteins as depicted by the anti-Flag antibody. (Ctrl refers to nuclear extracts from mock-transfected cells). B- EMSA was performed using equal amounts of the overexpressed NFATC1 proteins from HEK 293 cells transfected with Wt NFATC1 and NFATC1 mutants (P66L, I701L, P66L/I701L) and NFAT-consensus binding site as a probe. – ve sign indicates absence of nuclear extracts/* indicates NFATC1 monomer/** indicates NFATC1 Dimer/→ refers to the 32 P labeled free DNA probe. C- Quantification of the NFATC1 dimers in the EMSA using the TotalLab2010 software from Amersham shows a 30% decrease in DNA binding affinity of the single and double mutant as compared to the wild type NFATC1 protein.

Figure Legend Snippet: A- NFATC1 extracts from HEK 293 cells transfected with Wt NFATC1 and Mutants (P66L – I701L – P66L/I701L) were resolved on an SDS-PAGE prior to gel shift assays. Western blots showed equal amounts of expressed proteins as depicted by the anti-Flag antibody. (Ctrl refers to nuclear extracts from mock-transfected cells). B- EMSA was performed using equal amounts of the overexpressed NFATC1 proteins from HEK 293 cells transfected with Wt NFATC1 and NFATC1 mutants (P66L, I701L, P66L/I701L) and NFAT-consensus binding site as a probe. – ve sign indicates absence of nuclear extracts/* indicates NFATC1 monomer/** indicates NFATC1 Dimer/→ refers to the 32 P labeled free DNA probe. C- Quantification of the NFATC1 dimers in the EMSA using the TotalLab2010 software from Amersham shows a 30% decrease in DNA binding affinity of the single and double mutant as compared to the wild type NFATC1 protein.

Techniques Used: Transfection, SDS Page, Electrophoretic Mobility Shift Assay, Western Blot, Binding Assay, Labeling, Software, Mutagenesis

A- Wt NFATC1 or NFATC1 Mutants (P66L, I701L, P66L/I701L) were cotransfected with the human CCND1 promoter coupled luciferase reporter construct in the presence or absence of activated clacineurin (PPP3CA) in Hela cells. Six hours post transfection, media was changed and cells were harvested for luciferase assay after 36 hours. Relative luciferase activities are represented as fold activation. The data are the mean of three independent experiments done in duplicates +/− standard deviation. Significance (p<0.05) was assessed using the one-way Anova test. (* p<0.01, ** p<0.05) B- Wt NFATC1 or NFATC1 Mutants (P66L, I701L, P66L/I701L) were cotransfected with the human DEGS1 promoter coupled luciferase reporter construct in the presence or absence of activated clacineurin (PPP3CA) in Hela cells. Six hours post transfection, media was changed and cells were harvested for luciferase assay after 36 hours. Relative luciferase activities are represented as fold activation. The data are the mean of three independent experiments done in duplicates +/− standard deviation. Significance (p<0.05) was assessed using the one-way Anova test. (* p<0.01, ** p<0.05).

Figure Legend Snippet: A- Wt NFATC1 or NFATC1 Mutants (P66L, I701L, P66L/I701L) were cotransfected with the human CCND1 promoter coupled luciferase reporter construct in the presence or absence of activated clacineurin (PPP3CA) in Hela cells. Six hours post transfection, media was changed and cells were harvested for luciferase assay after 36 hours. Relative luciferase activities are represented as fold activation. The data are the mean of three independent experiments done in duplicates +/− standard deviation. Significance (p<0.05) was assessed using the one-way Anova test. (* p<0.01, ** p<0.05) B- Wt NFATC1 or NFATC1 Mutants (P66L, I701L, P66L/I701L) were cotransfected with the human DEGS1 promoter coupled luciferase reporter construct in the presence or absence of activated clacineurin (PPP3CA) in Hela cells. Six hours post transfection, media was changed and cells were harvested for luciferase assay after 36 hours. Relative luciferase activities are represented as fold activation. The data are the mean of three independent experiments done in duplicates +/− standard deviation. Significance (p<0.05) was assessed using the one-way Anova test. (* p<0.01, ** p<0.05).

Techniques Used: Luciferase, Construct, Transfection, Activation Assay, Standard Deviation

A- Wt NFATC1 or NFATC1 Mutants (P66L, I701L, P66L/I701L) were transfected with/without HAND2 and the DEGS1 promoter coupled to luciferase reporter construct in Hela cells. Six hours post transfection, media was changed and cells were harvested for luciferase assay after 36 hours. Relative luciferase activities are represented as fold activation. The data are the mean of three independent experiments done in duplicates +/− standard deviation. Wt NFATC1 and HAND2 synergistically activate DEGS1 promoter. This synergy was abrogated in all NFATC1 mutants. Significance (p<0.05) was assessed using the one-way Anova test. (* p<0.01, ** p<0.05) B- Wt NFATC1 or NFATC1 Mutants (P66L, I701L, P66L/I701L) were transfected with/without PPP3CA and with/without GATA5 to assess their combinatorial regulation of the DEGS1 promoter in HeLa cells. Six hours post transfection, media was changed and cells were harvested for luciferase assay after 36 hours. Relative luciferase activities are represented as fold activation. The data are the mean of three independent experiments done in duplicates +/− standard deviation. Wt NFATC1 cotransfected with GATA5 caused a synergistic activation of 35 fold, while transfection of Wt NFATC1 with PPP3CA and GATA5 caused even a stronger synergy reaching 68 fold. The synergestic activation was maintained in all mutants except for P66L/I701L double mutant where the synergy was totally lost. Significance (p<0.05) was assessed using the one-way Anova test. (* p<0.01, ** p<0.05).

Figure Legend Snippet: A- Wt NFATC1 or NFATC1 Mutants (P66L, I701L, P66L/I701L) were transfected with/without HAND2 and the DEGS1 promoter coupled to luciferase reporter construct in Hela cells. Six hours post transfection, media was changed and cells were harvested for luciferase assay after 36 hours. Relative luciferase activities are represented as fold activation. The data are the mean of three independent experiments done in duplicates +/− standard deviation. Wt NFATC1 and HAND2 synergistically activate DEGS1 promoter. This synergy was abrogated in all NFATC1 mutants. Significance (p<0.05) was assessed using the one-way Anova test. (* p<0.01, ** p<0.05) B- Wt NFATC1 or NFATC1 Mutants (P66L, I701L, P66L/I701L) were transfected with/without PPP3CA and with/without GATA5 to assess their combinatorial regulation of the DEGS1 promoter in HeLa cells. Six hours post transfection, media was changed and cells were harvested for luciferase assay after 36 hours. Relative luciferase activities are represented as fold activation. The data are the mean of three independent experiments done in duplicates +/− standard deviation. Wt NFATC1 cotransfected with GATA5 caused a synergistic activation of 35 fold, while transfection of Wt NFATC1 with PPP3CA and GATA5 caused even a stronger synergy reaching 68 fold. The synergestic activation was maintained in all mutants except for P66L/I701L double mutant where the synergy was totally lost. Significance (p<0.05) was assessed using the one-way Anova test. (* p<0.01, ** p<0.05).

Techniques Used: Transfection, Luciferase, Construct, Activation Assay, Standard Deviation, Mutagenesis

Figure Legend Snippet: Hypothetical pathway involving NFATC1 in endocardial cushion proliferation and valve maturation.

Techniques Used:

Comparison:

Article Title: Nuclear factor of activated T cells 2 is required for osteoclast differentiation and function in vitro but not in vivo.
Article Snippet: Equal amounts of RNA were reverse‐transcribed using the iScript RT‐PCR kit (BioRad) and amplified in the presence of specific primers (all primers from Integrated DNA Technologies (IDT), Coralville, IA; Table 2) with the SsoAdvanced Universal SYBR Green Supermix (BioRad) at 60°C for 40 cycles. .. Transcript copy number was estimated by comparison with a serial dilution of cDNA for Nfatc1 (Addgene plasmid 11793), Nfatc2 (Addgene plasmid 11791, both created by A Rao, La Jolla, CA), Acp5, Ctsk, and Calcr (Thermo Fisher Scientific). .. Tnfrsf11a copy number was estimated by comparison to a serial dilution of a 152 base pair (bp) synthetic DNA template (IDT) cloned into pcDNA3.1(−) (Thermo Fisher Scientific) by isothermal single reaction assembly using commercially available reagents (New England BioLabs, Ipswich, MA).

Article Title: NUCLEAR FACTOR OF ACTIVATED T CELLS 2 IS REQUIRED FOR OSTEOCLAST DIFFERENTIATION AND FUNCTION IN VITRO BUT NOT IN VIVO
Article Snippet: Equal amounts of RNA were reverse-transcribed using the iScript RT-PCR kit (BioRad) and amplified in the presence of specific primers (all primers from Integrated DNA Technologies, IDT, Coralville, IA; ) with the SsoAdvanced TM Universal SYBR Green Supermix (BioRad) at 60°C for 40 cycles. .. Transcript copy number was estimated by comparison with a serial dilution of cDNA for Nfatc1 (Addgene plasmid 11793), Nfatc2 (Addgene plasmid 11791, both created by A. Rao, La Jolla, CA), Acp5 , Ctsk and Calcr (Thermo Fisher Scientific). ..

Serial Dilution:

Plasmid Preparation:

Image Search Results

Journal: PLoS ONE

Article Title: Two Heterozygous Mutations in NFATC1 in a Patient with Tricuspid Atresia

doi: 10.1371/journal.pone.0049532

Figure Lengend Snippet: A- The missense SNPs lead to a P66L substitution at the N-terminal region of the protein near the calcineurin-docking site (Cln binding), and to a I701L substitution at the C-terminal region downstream of the Rel Homolgy Domain (RHD). The schematic represents isoform A, the most abundant NFATC1 protein with 717 amino acids, a transactivation domain (TAD) at the N-terminus, and a DNA binding domain at the C-terminus. (NLS = nuclear localization signal, NES = nuclear export signal, and SP = Serine-Proline). B- The NFATC1 secondary structures were predicted and visualized by the Discovery Studio program (Acclerys Inc.). The results demonstrate the formation of a new beta-sheet in the P66L mutant and a deletion of a beta-sheet in the I701L mutant as compared to the wild type protein.

Article Snippet: After identifying each mutant gene sequence, an oligonucleotide (forward primer) harboring the desired mutation was synthesized in a way to complement the human NFATC1 cDNA (Addgene) subcloned in the pCEP4 expression vector (Invitrogen).

Techniques: Binding Assay, Mutagenesis

Journal: PLoS ONE

Article Title: Two Heterozygous Mutations in NFATC1 in a Patient with Tricuspid Atresia

doi: 10.1371/journal.pone.0049532

Figure Lengend Snippet: Frequency of the NFATC1 mutations according to the Exome Sequencing Project (ESP).

Techniques: Sequencing

Journal: PLoS ONE

Article Title: Two Heterozygous Mutations in NFATC1 in a Patient with Tricuspid Atresia

doi: 10.1371/journal.pone.0049532

Figure Lengend Snippet: A- Immunofluorescence of HeLa cells transfected with plasmids encoding for the Wt NFATC1 and NFATC1 Mutants (P66L, I701L, P66L/I701L). The localization of NFATC1 was visualized using an anti-Flag antibody. Nuclei of cells were visualized using the Hoechst dye (blue color). Wt and NFATC1 mutants localized to the cytoplasm in the absence of PPP3CA (red color). (Magnification ×40). B- Immunofluorescence of HeLa cells transfected with plasmids encoding for the Wt NFATC1 and NFATC1 Mutants (P66L, I701L, P66L/I701L) co-transfected with PP3CA. The localization of NFATC1 was visualized using an anti-Flag antibody (red color) while PP3CA was visualized using anti-HA antibody (green color). Nuclei of cells were visualized using Hoechst dye (blue color). Most of the cells co-transfected with the double NFATC1 mutant were retained in the cytoplasm around the nuclear membrane, whereas in the other cases, the protein was totally translocated to the nucleus. (Magnification ×40). Yellow arrows indicate cytoplasmic (peri-nuclear) staining, while red arrows indicate nuclear staining.

Techniques: Immunofluorescence, Transfection, Mutagenesis, Staining

Journal: PLoS ONE

Article Title: Two Heterozygous Mutations in NFATC1 in a Patient with Tricuspid Atresia

doi: 10.1371/journal.pone.0049532

Figure Lengend Snippet: A- NFATC1 extracts from HEK 293 cells transfected with Wt NFATC1 and Mutants (P66L – I701L – P66L/I701L) were resolved on an SDS-PAGE prior to gel shift assays. Western blots showed equal amounts of expressed proteins as depicted by the anti-Flag antibody. (Ctrl refers to nuclear extracts from mock-transfected cells). B- EMSA was performed using equal amounts of the overexpressed NFATC1 proteins from HEK 293 cells transfected with Wt NFATC1 and NFATC1 mutants (P66L, I701L, P66L/I701L) and NFAT-consensus binding site as a probe. – ve sign indicates absence of nuclear extracts/* indicates NFATC1 monomer/** indicates NFATC1 Dimer/→ refers to the 32 P labeled free DNA probe. C- Quantification of the NFATC1 dimers in the EMSA using the TotalLab2010 software from Amersham shows a 30% decrease in DNA binding affinity of the single and double mutant as compared to the wild type NFATC1 protein.

Techniques: Transfection, SDS Page, Electrophoretic Mobility Shift Assay, Western Blot, Binding Assay, Labeling, Software, Mutagenesis

Journal: PLoS ONE

Article Title: Two Heterozygous Mutations in NFATC1 in a Patient with Tricuspid Atresia

doi: 10.1371/journal.pone.0049532

Figure Lengend Snippet: A- Wt NFATC1 or NFATC1 Mutants (P66L, I701L, P66L/I701L) were cotransfected with the human CCND1 promoter coupled luciferase reporter construct in the presence or absence of activated clacineurin (PPP3CA) in Hela cells. Six hours post transfection, media was changed and cells were harvested for luciferase assay after 36 hours. Relative luciferase activities are represented as fold activation. The data are the mean of three independent experiments done in duplicates +/− standard deviation. Significance (p<0.05) was assessed using the one-way Anova test. (* p<0.01, ** p<0.05) B- Wt NFATC1 or NFATC1 Mutants (P66L, I701L, P66L/I701L) were cotransfected with the human DEGS1 promoter coupled luciferase reporter construct in the presence or absence of activated clacineurin (PPP3CA) in Hela cells. Six hours post transfection, media was changed and cells were harvested for luciferase assay after 36 hours. Relative luciferase activities are represented as fold activation. The data are the mean of three independent experiments done in duplicates +/− standard deviation. Significance (p<0.05) was assessed using the one-way Anova test. (* p<0.01, ** p<0.05).

Techniques: Luciferase, Construct, Transfection, Activation Assay, Standard Deviation

Journal: PLoS ONE

Article Title: Two Heterozygous Mutations in NFATC1 in a Patient with Tricuspid Atresia

doi: 10.1371/journal.pone.0049532

Figure Lengend Snippet: A- Wt NFATC1 or NFATC1 Mutants (P66L, I701L, P66L/I701L) were transfected with/without HAND2 and the DEGS1 promoter coupled to luciferase reporter construct in Hela cells. Six hours post transfection, media was changed and cells were harvested for luciferase assay after 36 hours. Relative luciferase activities are represented as fold activation. The data are the mean of three independent experiments done in duplicates +/− standard deviation. Wt NFATC1 and HAND2 synergistically activate DEGS1 promoter. This synergy was abrogated in all NFATC1 mutants. Significance (p<0.05) was assessed using the one-way Anova test. (* p<0.01, ** p<0.05) B- Wt NFATC1 or NFATC1 Mutants (P66L, I701L, P66L/I701L) were transfected with/without PPP3CA and with/without GATA5 to assess their combinatorial regulation of the DEGS1 promoter in HeLa cells. Six hours post transfection, media was changed and cells were harvested for luciferase assay after 36 hours. Relative luciferase activities are represented as fold activation. The data are the mean of three independent experiments done in duplicates +/− standard deviation. Wt NFATC1 cotransfected with GATA5 caused a synergistic activation of 35 fold, while transfection of Wt NFATC1 with PPP3CA and GATA5 caused even a stronger synergy reaching 68 fold. The synergestic activation was maintained in all mutants except for P66L/I701L double mutant where the synergy was totally lost. Significance (p<0.05) was assessed using the one-way Anova test. (* p<0.01, ** p<0.05).

Techniques: Transfection, Luciferase, Construct, Activation Assay, Standard Deviation, Mutagenesis

Journal: PLoS ONE

Article Title: Two Heterozygous Mutations in NFATC1 in a Patient with Tricuspid Atresia

doi: 10.1371/journal.pone.0049532

Figure Lengend Snippet: Hypothetical pathway involving NFATC1 in endocardial cushion proliferation and valve maturation.

Techniques:

(a) Pedigree of the family involved in the study. Black arrow represents the proband who carried tricuspid atresia. (b) Sanger sequencing confirmed that the proband carried a NFATC1 mutation which c.964G changed to A. (c and d) Fetal echocardiography of the proband indicated right ventricular dysplasia and tricuspid atresia

Journal: Molecular Genetics & Genomic Medicine

Article Title: Genetic and functional analyses detect one pathological NFATC1 mutation in a Chinese tricuspid atresia family.

doi: 10.1002/mgg3.1771

Figure Lengend Snippet: (a) Pedigree of the family involved in the study. Black arrow represents the proband who carried tricuspid atresia. (b) Sanger sequencing confirmed that the proband carried a NFATC1 mutation which c.964G changed to A. (c and d) Fetal echocardiography of the proband indicated right ventricular dysplasia and tricuspid atresia

Article Snippet: pCMV3.1‐NFATC1 expression vector containing human NFATC1 coding sequence was purchased from Sino Biological (catalog: HG13963‐CF).

Techniques: Sequencing, Mutagenesis

Detailed information of candidate gene mutations

Journal: Molecular Genetics & Genomic Medicine

Article Title: Genetic and functional analyses detect one pathological NFATC1 mutation in a Chinese tricuspid atresia family.

doi: 10.1002/mgg3.1771

Figure Lengend Snippet: Detailed information of candidate gene mutations

Article Snippet: pCMV3.1‐NFATC1 expression vector containing human NFATC1 coding sequence was purchased from Sino Biological (catalog: HG13963‐CF).

Techniques: Software

Functional experiments of NFATC1. (a) Image of western blotting. (b) Analysis of gray value of western blotting, ** p < 0.01 (c) Relative luciferase activity of DEGS1 promoter raised by NFATC1‐WT, NFATC1‐p.D322N, and control group. ** p < 0.01

Journal: Molecular Genetics & Genomic Medicine

Article Title: Genetic and functional analyses detect one pathological NFATC1 mutation in a Chinese tricuspid atresia family.

doi: 10.1002/mgg3.1771

Figure Lengend Snippet: Functional experiments of NFATC1. (a) Image of western blotting. (b) Analysis of gray value of western blotting, ** p < 0.01 (c) Relative luciferase activity of DEGS1 promoter raised by NFATC1‐WT, NFATC1‐p.D322N, and control group. ** p < 0.01

Article Snippet: pCMV3.1‐NFATC1 expression vector containing human NFATC1 coding sequence was purchased from Sino Biological (catalog: HG13963‐CF).

Techniques: Functional Assay, Western Blot, Luciferase, Activity Assay

Primers used for qRT-PCR determinations. GenBank accession numbers identify transcript recognized by primer pairs.

Journal: Journal of cellular biochemistry

Article Title: NUCLEAR FACTOR OF ACTIVATED T CELLS 2 IS REQUIRED FOR OSTEOCLAST DIFFERENTIATION AND FUNCTION IN VITRO BUT NOT IN VIVO

doi: 10.1002/jcb.27212

Figure Lengend Snippet: Primers used for qRT-PCR determinations. GenBank accession numbers identify transcript recognized by primer pairs.

Article Snippet: Transcript copy number was estimated by comparison with a serial dilution of cDNA for Nfatc1 (Addgene plasmid 11793), Nfatc2 (Addgene plasmid 11791, both created by A. Rao, La Jolla, CA), Acp5 , Ctsk and Calcr (Thermo Fisher Scientific).

Techniques: Sequencing

BMMs derived from 1 month old Lyz2Cre/WT;Nfatc2Δ/Δ mice and Nfatc2loxP/loxP control littermates were cultured for 4 days in the presence of M-CSF at 30 ng/ml and of RANKL at 10 ng/ml. The cells were collected at the indicated times for extraction of total RNA and proteins. (A) Nfatc2 and Nfatc1 mRNA levels were measured by qRT-PCR. Transcript levels are reported as copy number corrected for Rpl38 mRNA levels. Values are means ± SD; n = 5 control and n = 3 Nfatc2Δ/Δ biological replicates. Two technical replicates were used for each qRT-PCR reaction. *Significantly different between Nfatc2Δ/Δ and control, p < 0.05. (B) 50 μg of total protein were separated by SDS-PAGE and NFATc1 and NFATc2 levels were detected by using immunoblot anti-NFATc1 and anti-NFATc2 antibodies, respectively. β-Actin levels served as a loading control in the same blot. (C) Tnfrs11a, Acp5, Ctsk and Calcr mRNA levels in total RNA were measured by qRT-PCR. Transcript levels are reported as copy number corrected for Rpl38. Values are means ± SD; n = 5 control and n = 3 Nfatc2Δ/Δ biological replicates. Two technical replicates were used for each qRT-PCR reaction. *Significantly different between Nfatc2Δ/Δ and control, p < 0.05.

Journal: Journal of cellular biochemistry

Article Title: NUCLEAR FACTOR OF ACTIVATED T CELLS 2 IS REQUIRED FOR OSTEOCLAST DIFFERENTIATION AND FUNCTION IN VITRO BUT NOT IN VIVO

doi: 10.1002/jcb.27212

Figure Lengend Snippet: BMMs derived from 1 month old Lyz2Cre/WT;Nfatc2Δ/Δ mice and Nfatc2loxP/loxP control littermates were cultured for 4 days in the presence of M-CSF at 30 ng/ml and of RANKL at 10 ng/ml. The cells were collected at the indicated times for extraction of total RNA and proteins. (A) Nfatc2 and Nfatc1 mRNA levels were measured by qRT-PCR. Transcript levels are reported as copy number corrected for Rpl38 mRNA levels. Values are means ± SD; n = 5 control and n = 3 Nfatc2Δ/Δ biological replicates. Two technical replicates were used for each qRT-PCR reaction. *Significantly different between Nfatc2Δ/Δ and control, p < 0.05. (B) 50 μg of total protein were separated by SDS-PAGE and NFATc1 and NFATc2 levels were detected by using immunoblot anti-NFATc1 and anti-NFATc2 antibodies, respectively. β-Actin levels served as a loading control in the same blot. (C) Tnfrs11a, Acp5, Ctsk and Calcr mRNA levels in total RNA were measured by qRT-PCR. Transcript levels are reported as copy number corrected for Rpl38. Values are means ± SD; n = 5 control and n = 3 Nfatc2Δ/Δ biological replicates. Two technical replicates were used for each qRT-PCR reaction. *Significantly different between Nfatc2Δ/Δ and control, p < 0.05.

Techniques: Derivative Assay, Control, Cell Culture, Extraction, Quantitative RT-PCR, SDS Page, Western Blot

P-S induces the expression of NFATc1 in pancreatic cancer cells. (A) BxPC-3 and Mia PaCa-2 cells were transfected with NFATc1 shRNA or cDNA, clones selected and relative mRNA levels analyzed. (B) BxPC-3 NFATc1 WT and knockdown (Kd) cells were treated with vehicle, P-S 0.5 and 1.0×IC 50 , or sulindac 1.0×IC 50 for 24 h. Cells were lysed and NFATc1 expression is analyzed by western blotting. (C) Mia PaCa-2 NFATc1 WT, Kd and overexpressing (Ov.) cells were treated with vehicle, P-S 0.5 and 1.0×IC 50 , or sulindac 1.0×IC 50 for 24 h. Cells were lysed and NFATc1 expression is analyzed by western blotting. * p<0.05, compared to control group.

Journal: International Journal of Oncology

Article Title: Phospho-sulindac inhibits pancreatic cancer growth: NFATc1 as a drug resistance candidate

doi: 10.3892/ijo.2013.2190

Figure Lengend Snippet: P-S induces the expression of NFATc1 in pancreatic cancer cells. (A) BxPC-3 and Mia PaCa-2 cells were transfected with NFATc1 shRNA or cDNA, clones selected and relative mRNA levels analyzed. (B) BxPC-3 NFATc1 WT and knockdown (Kd) cells were treated with vehicle, P-S 0.5 and 1.0×IC 50 , or sulindac 1.0×IC 50 for 24 h. Cells were lysed and NFATc1 expression is analyzed by western blotting. (C) Mia PaCa-2 NFATc1 WT, Kd and overexpressing (Ov.) cells were treated with vehicle, P-S 0.5 and 1.0×IC 50 , or sulindac 1.0×IC 50 for 24 h. Cells were lysed and NFATc1 expression is analyzed by western blotting. * p<0.05, compared to control group.

Article Snippet: The NFATc1 Mia PaCa-2 overexpression cell line was generated using NFATc1 human cDNA clone from Origene (Rockville, MD, USA).

Techniques: Expressing, Transfection, shRNA, Clone Assay, Knockdown, Western Blot, Control

P-S induces nuclear NFATc1 expression and its downstream signaling. (A) BxPC-3 NFATc1 WT and knockdown (Kd) cells were treated with P-S for 24 h, lysed and nuclear NFATc1 was determined by western blotting. (B) Mia PaCa-2 NFATc1 WT, Kd and overexpressing (Ov.) cells were treated with P-S for 24 h, lysed and nuclear NFATc1 was determined by western blotting. (C, D and E) BxPC-3 NFATc1 WT and Kd cells were treated with vehicle, P-S 0.5 and 1.0×IC 50 , or sulindac 1.0×IC 50 for 24 h. Cells were lysed and the protein expression of COX-2, c-Myc, pAKT and pERK were analyzed by western blotting. * p<0.05, compared to control group.

Journal: International Journal of Oncology

Article Title: Phospho-sulindac inhibits pancreatic cancer growth: NFATc1 as a drug resistance candidate

doi: 10.3892/ijo.2013.2190

Figure Lengend Snippet: P-S induces nuclear NFATc1 expression and its downstream signaling. (A) BxPC-3 NFATc1 WT and knockdown (Kd) cells were treated with P-S for 24 h, lysed and nuclear NFATc1 was determined by western blotting. (B) Mia PaCa-2 NFATc1 WT, Kd and overexpressing (Ov.) cells were treated with P-S for 24 h, lysed and nuclear NFATc1 was determined by western blotting. (C, D and E) BxPC-3 NFATc1 WT and Kd cells were treated with vehicle, P-S 0.5 and 1.0×IC 50 , or sulindac 1.0×IC 50 for 24 h. Cells were lysed and the protein expression of COX-2, c-Myc, pAKT and pERK were analyzed by western blotting. * p<0.05, compared to control group.

Article Snippet: The NFATc1 Mia PaCa-2 overexpression cell line was generated using NFATc1 human cDNA clone from Origene (Rockville, MD, USA).

Techniques: Expressing, Knockdown, Western Blot, Control

Effect of NFATc1 knockdown or overexpression on 24-h IC 50 values of P-S in BxPC-3 and MIA PaCa-2 cells.

Journal: International Journal of Oncology

Article Title: Phospho-sulindac inhibits pancreatic cancer growth: NFATc1 as a drug resistance candidate

doi: 10.3892/ijo.2013.2190

Figure Lengend Snippet: Effect of NFATc1 knockdown or overexpression on 24-h IC 50 values of P-S in BxPC-3 and MIA PaCa-2 cells.

Article Snippet: The NFATc1 Mia PaCa-2 overexpression cell line was generated using NFATc1 human cDNA clone from Origene (Rockville, MD, USA).

Techniques: Knockdown, Over Expression, Transfection

Effect of NFATc1 knockdown on 24-h IC 50 values of chemotherapeutic drugs in BxPC-3 cells.

Journal: International Journal of Oncology

Article Title: Phospho-sulindac inhibits pancreatic cancer growth: NFATc1 as a drug resistance candidate

doi: 10.3892/ijo.2013.2190

Figure Lengend Snippet: Effect of NFATc1 knockdown on 24-h IC 50 values of chemotherapeutic drugs in BxPC-3 cells.

Article Snippet: The NFATc1 Mia PaCa-2 overexpression cell line was generated using NFATc1 human cDNA clone from Origene (Rockville, MD, USA).

Techniques: Knockdown, Transfection

NFATc1 modulates the anticancer effect of P-S and chemotherapeutic drugs. (A) Efficacy of P-S in mice bearing Mia PaCa-2 wild-type (WT), NFATc1-knockdown (Kd), or NFATc1 overexpressing xenografts. Mice were treated with vehicle or P-S 100 mg/kg/day. (B) BxPC-3 NFATc1 WT and Kd cells were treated with vehicle, P-S 0.5×IC 50 + CsA and 1.0×IC 50 + CsA, or CsA alone for 24 h. NFATc1 protein expression was analyzed by western blotting. * p<0.05, compared to control group.

Journal: International Journal of Oncology

Article Title: Phospho-sulindac inhibits pancreatic cancer growth: NFATc1 as a drug resistance candidate

doi: 10.3892/ijo.2013.2190

Figure Lengend Snippet: NFATc1 modulates the anticancer effect of P-S and chemotherapeutic drugs. (A) Efficacy of P-S in mice bearing Mia PaCa-2 wild-type (WT), NFATc1-knockdown (Kd), or NFATc1 overexpressing xenografts. Mice were treated with vehicle or P-S 100 mg/kg/day. (B) BxPC-3 NFATc1 WT and Kd cells were treated with vehicle, P-S 0.5×IC 50 + CsA and 1.0×IC 50 + CsA, or CsA alone for 24 h. NFATc1 protein expression was analyzed by western blotting. * p<0.05, compared to control group.

Article Snippet: The NFATc1 Mia PaCa-2 overexpression cell line was generated using NFATc1 human cDNA clone from Origene (Rockville, MD, USA).

Techniques: Knockdown, Expressing, Western Blot, Control

Proposed mechanistic interaction of P-S and NFATc1. PS induces ROS, followed by the dephosphorylation of NFATc1, which in turn, leads to increased cytoplasmic and nuclear NFATc1 protein expression. Activation of NFATc1-mediated transcription increased the expression of c-Myc and COX-2. When PS is combined with CsA, the latter reduces the induction of nuclear and cytoplasmic NFATc1 protein expression. CsA can be conceptualized in this diagram as affecting calcium homeostasis, which in turn modulates the activity of NFATc1. Thus, PS and CsA may synergize to inhibit pancreatic carcinogenesis.

Journal: International Journal of Oncology

Article Title: Phospho-sulindac inhibits pancreatic cancer growth: NFATc1 as a drug resistance candidate

doi: 10.3892/ijo.2013.2190

Figure Lengend Snippet: Proposed mechanistic interaction of P-S and NFATc1. PS induces ROS, followed by the dephosphorylation of NFATc1, which in turn, leads to increased cytoplasmic and nuclear NFATc1 protein expression. Activation of NFATc1-mediated transcription increased the expression of c-Myc and COX-2. When PS is combined with CsA, the latter reduces the induction of nuclear and cytoplasmic NFATc1 protein expression. CsA can be conceptualized in this diagram as affecting calcium homeostasis, which in turn modulates the activity of NFATc1. Thus, PS and CsA may synergize to inhibit pancreatic carcinogenesis.

Article Snippet: The NFATc1 Mia PaCa-2 overexpression cell line was generated using NFATc1 human cDNA clone from Origene (Rockville, MD, USA).

Techniques: De-Phosphorylation Assay, Expressing, Activation Assay, Activity Assay

Review

human nfatc1 cdna (Addgene inc)

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1) Product Images from "Two Heterozygous Mutations in NFATC1 in a Patient with Tricuspid Atresia"

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