MA3-024

antibody from Invitrogen Antibodies

Targeting: NFATC1 NF-ATC, NFAT2, NFATc

Provider product page for MA3-024

Western blot

Immunocytochemistry

Immunohistochemistry

Other assay

Antibody data

Antibody Data
Antigen structure
References [91]
Comments [0]
Validations
Western blot [1]
Immunocytochemistry [3]
Immunohistochemistry [3]
Other assay [40]

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Product number: MA3-024 - Provider product page
Provider: Invitrogen Antibodies
Product name: NFATC1 Monoclonal Antibody (7A6)
Antibody type: Monoclonal
Antigen: Other
Description: MA3-024 detects NFATc1 (NFAT2) from transfected human, rat, mouse, and monkey cells. This antibody does not cross react with NFATc2 (NFAT1).
Antibody clone number: 7A6
Concentration: Conc. Not Determined

NFATC1 protein structure - MA3-024 shown in red.

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Experimental details: Immunofluorescent analysis of NFATc1 using NFATc1 Monoclonal Antibody (7A6) (Product # MA3-024) shows staining in 293Cells. NFATc1 (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with an antibody recognizing NFATc1 (Product # MA3-024) at a dilution of 1:20 over night at 4 °C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35552 for GAR, Product # 35503 for GAM). Images were taken at 60X magnification.

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Experimental details: Immunofluorescent analysis of NFATc1 using NFATc1 Monoclonal Antibody (7A6) (Product # MA3-024) shows staining in Hela Cells. NFATc1 (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with an antibody recognizing NFATc1 (Product # MA3-024) at a dilution of 1:20 over night at 4 °C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35552 for GAR, Product # 35503 for GAM). Images were taken at 60X magnification.

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Experimental details: Immunofluorescent analysis of NFATc1 using NFATc1 Monoclonal Antibody (7A6) (Product # MA3-024) shows staining in MCF-7 Cells. NFATc1 (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with an antibody recognizing NFATc1 (Product # MA3-024) at a dilution of 1:20 over night at 4 °C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35552 for GAR, Product # 35503 for GAM). Images were taken at 60X magnification.

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Experimental details: Immunohistochemistry was performed on normal biopsies of deparaffinized Human skeletal muscle tissue. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:20 with a mouse monoclonal antibody recognizing NFATc1 (Product # MA3-024) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

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Experimental details: Immunohistochemistry was performed on normal biopsies of deparaffinized Human spleen tissue. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:100 with a mouse monoclonal antibody recognizing NFATc1 (Product # MA3-024) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

Supportive validation

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Experimental details: Immunohistochemistry was performed on normal biopsies of deparaffinized Human tonsil tissue. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:200 with a mouse monoclonal antibody recognizing NFATc1 (Product # MA3-024) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

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Experimental details: Figure 2 Isx9 increased NFAT transcriptional activity. ( A ) Dose-dependent activation of the NFAT reporter in beta cells after 24 h treatment with increasing dose of Isx9 in the presence or absence of 0.3 uM FK506, mean +- SEM of three independent experiments in triplicates, # p < 0.05 and ## p < 0.01 Isx9 versus non-treated cells; * p < 0.05, ** p < 0.01 effect of FK506 treatment versus control for each Isx9 dose. ( B ) Immunoblotting of NFATc1 and D28K in MIN6 whole cell extract after 48 h treatment with vehicle DMSO (Veh) or 10 uM Isx9 in the presence or absence of calcineurin inhibitor FK506. ( C ) Subcellular fractionation (Pierce) of MIN6 cells treated with Isx9 or vehicle into cytoplasmic (Cyt), nuclear (NE) and membrane (Mbr) fractions followed by immunoblotting of NFATc1 and D28K. alpha-Tubulin, Nkx6.1, and Transferrin receptors are used as loading controls. ( D ) Calcineurin activity in MIN6 represented as % of untreated cells treated with increasing doses of Isx9, FK506 is used as a negative control, mean +- SEM of three independent experiments in triplicates, ** p < 0.01 vs. control. ( E ) Immunoblotting of phospho-Creb1-Ser 133, D28K, and GAPDH after increasing dose of Isx9 for 24 h or ( F ) after 8 h and 24 h treatment with 10 uM Isx9 in MIN6 cells.

Supportive validation

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Experimental details: Figure 1. p38 Alternative activation is required for TCR-induced expression of NFATc1 and IRF4 in CD4 + T cells. (A) Purified CD4 + T cells were stimulated with anti-CD3/CD28 in the presence or absence of SB203580 and immunoblotted for the indicated transcription factors 48 h later. (B and C) Purified CD4 + T cells were stimulated with anti-CD3/CD28 in the presence of 11R-VIVIT or 11R-VEET. Irf4 mRNA (B) and protein (C) were determined 24 h later. (D and E) Purified CD4 + T cells from WT and DKI mice were stimulated with anti-CD3/CD28 or PMA plus ionomycin, as indicated for the indicated times and analyzed for expression of Nfatc1 and Irf4 mRNA (D) and protein (E). Results shown in A-E are representative of three independent experiments each. (F) Naive CD4 + T cells were stimulated with either anti-CD3/CD28 or PMA and ionomycin under neutral or Th17-skewing conditions for 3 d. IL-17A and IFN-gamma expression were determined by intracellular staining and flow cytometry after restimulation with PMA and ionomycin. Results shown in the left panel are representative of three independent experiments and bar graphs in the right panel show the mean +- SEM of all three. *, P < 0.05 (unpaired two-tailed Student's t test).

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Experimental details: Figure 3. MAPK-cascade activated p38 inhibits functions distal to alternatively activated p38. (A) Purified CD4 + T cells were cultured overnight without stimulation, and then treated with either UV (50 J/m 2 or 100 J/m 2 or 200 J/m 2 ), 0.6 M sorbitol for 30 min, or PMA (10 ng/ml) and ionomycin (1 ug/ml) for 1 h. Cell lysates were immunoblotted with anti-phospho-p38. The results are representative of three independent experiments. (B) Purified CD4 + T cells were treated with either 0.6 M sorbitol for 30 min, 50 J/m 2 UV, or PMA and ionomycin were stimulated with anti-CD3/CD28 for 24 h. The expression of Nfatc1 and Irf4 was determined by quantitative real-time PCR. Results are the mean +- SEM of three independent experiments. *, P < 0.05 (unpaired two-tailed Student's t test). (C) Cells were treated as in B, and IRF4 expression was determined by Western blot. The results are representative of two independent experiments. (D) Freshly purified T cells were stimulated or not with anti-CD3/CD28 for 48 h. Cytosolic and nuclear fractions were separated in a low percentage SDS-PAGE (8%) gel and immunoblotted for NFATc1. The results are representative of three independent experiments. (E) CD4 + T cells were stimulated for 48 h with anti-CD3/CD28, and then treated with sorbitol in the presence or absence of SB203580 and/or SP600125 for 30 min, rested for 1 h, then immunoblotted for NFATc1 in cytosolic and nuclear fractions. Results are representative of three independent experime

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Experimental details: Figure 4 GDF11 activates Smad2/3-dependent TGF-beta pathway. ( a ) Gene expression levels of the BMMs stimulated without or with 100 ng ml -1 rGDF11 for 24 h (three biological replicates per group). 467 genes (red) were upregulated and 679 genes (black) were downregulated. 100 ng ml -1 M-CSF was present in all settings. ( b ) Heatmap of the osteoclastogenesis associated genes. ( c ) Quantitative RT-PCR confirmed the increased expression of osteoclast key marker genes. Results are shown as mean+-s.d.; n =3. *** P

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Experimental details: Fig 1 DYRK1A increased NFATc1 protein expression and its nuclear translocation. A. HEK293 cells were transfected with p-NFATc1mycFLAG and pCMV-DYRK1A or pGFP-V-RS-shDYRK1A. NFATc1 expression was detected by mouse anti-FLAG antibody immunoblotting. DYRK1A was blotted with anti-DYRK1A antibody. B. Endogenous NFATc1 was detected by anti-NFATc1 antibody in HEK293 cells transfected with pCMV-DYRK1A. An activated Jurkat T cells treated with 5uM ionomycin plus 500uM calcium chloride for 12 hours was used as positive control. C. HEK293 cells were co-transfected with pWT-NFATc2 and pCMV-DYRK1A. Anti-HA antibody was used to detect NFATc2. D and E. HEK293 cells were co-transfected with p-NFATc1mycFLAG and pCMV-DYRK1A orpGFP-V-RS-shDYRK1A. Mouse anti-FLAG antibody was used to detect NFATc1 protein in nucleus or cytoplasm.Beta-actin was used as cytoplasmic control and TBP was used as nuclear protein loading control. F. HEK293 cells were treated with ionomycin. Mouse anti-FLAG antibody was used to detect NFATc1 protein in nucleus or cytoplasm beta-actin was used as cytoplasmic control and TBP was used as nuclear protein loading control. Values represent means+-SD, n = 3. DY1A means DYRK1A. shDY1A means shDYRK1A.

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Experimental details: Fig 4 Identification of DYRK1A-targeted motifs in NFATc1 protein. A. Diagram showed NFATs sequence alignment. Sequences of NFATc1 (NP_765978), NFATc2 (NP_775114), NFATc3 (NP_775118), and NFATc4 (NP_001129494) were aligned using ClustalW2 software. Conserved regions among NFATs are underlined. Conserved serine residues in four NFATs are shown in blue. The DYRK1A target motifs are labeled as yellow highlights. The DYRK1A phosphorylation serine residues were in red and bold. B. Schematic diagram represents the domain structure of NFATc1 and location of five deletion constructs. AD: activation domain. SPRIEIT: calcineurin binding motif. SP: sp repeats region; SRR: serine-rich region; NLS: nuclear localization sequence. C. Five deletion constructs of NFATc1 were co-transfected with pCMV-DYRK1A in HEK293 cells. D. Deletionconstructs of NFATc1 was co-transfected with pCMV-DYRK1A in HEK293 cells. Anti-DYRK1A antibody was used as the immunoprecipitation antibody, and anti-FLAG antibody was used to detect NFATc1 in immunoblotting.

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Experimental details: Figure 5 Immunofluorescence images of GSK-3 beta ((a) red: nucleus; green: GSK-3 beta ) and beta -catenin ((b) blue: nucleus; green: beta -catenin). Scale bar = 200 mu m.

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Experimental details: Figure 6 Immunofluorescence images of RANKL ((a) red: nucleus; light blue: RANKL) and NFATc1 ((b) blue: nucleus; light blue: NFATc1). Scale bar = 200 mu m.

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Experimental details: Usp34 knockdown results in enhanced osteoclast differentiation of RAW264.7 cells. ( A ) qRT-PCR analysis of the expression of Usp34 during osteoclast induction of RAW264.7 cells. n = 3. ( B ) qRT-PCR and ( C ) Western blot confirmed successful knockdown of Usp34 in RAW264.7 cells. n = 3. ( D ) Representative images of TRAP staining. RAW264.7 cells were transfected with siRNA and stimulated with 50 ng/mL RANKL for 3 days. Scale bar = 100 mum. ( E ) Quantification of osteoclast number and ( F ) quantification of osteoclast size. No less than three nuclei in TRAP-positive cells were counted as osteoclasts. n = 6. ( G ) qRT-PCR detected the mRNA expression of osteoclast markers in the presence of RANKL for 2 days. n = 3. ( H ) Western blot analysis of indicated osteoclast markers after 2-day osteoclast induction. All quantified data are presented by box-plot with median value (cross), interquartile range (box), minimum/maximum (whiskers), and symbols representing all data points. The p values were calculated by one-way ANOVA with Tukey's post hoc test ( A ) or two-tailed Student's t test ( B , E , F , G ).

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Experimental details: Loss of USP34 increases osteoclastogenesis in vitro. ( A ) TRAP staining of multinucleated cells. Scale bar = 100 mum. ( B , C ) Quantification of osteoclasts number and size after 6-day osteoclast induction. n = 6. ( D ) Representative images of bone resorption pits after 7-day M-CSF (50 ng/mL) and RANKL (50 ng/mL) treatment. Scale bar = 100 mum. ( E , F ) Quantification of pits number and pits area after osteoclast induction. n = 6. ( G ) TRAP staining after BMMs and calvarial osteoblasts coculture. Cells were cultured with 1,25-(OH) 2 D 3 and PGE2 for 5 days. Scale bar = 100 mum. ( H , I ) Quantification of osteoclasts number and size after 5-day induction. n = 6. ( J ) qRT-PCR analysis of osteoclast markers for the indicated time periods. n = 3. ( K ) BMMs were treated with RANKL for 2 days and immunoblotted with indicated antibodies. All quantified data are presented by box-plot with median value (cross), interquartile range (box), minimum/maximum (whiskers), and symbols representing all data points. The p values were calculated by two-tailed Student's t test.

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Experimental details: FIGURE 4 Administration of VEGF-C 156S improves cardiac lymphangiogenesis and edema after pressure overload. (A) Schematic of the methods for treatment of WT mice with saline or VEGF-C 156S at doses of 33 (VEGF-C -L ) and 100 (VEGF-C -H ) ng/g and TAC for 6 weeks. (B) Heart sections stained with an anti-LYVE-1 (red) or anti-VEGFR-3 antibody (green) (left, scale bar: 50 mum) and quantification of LYVE-1 + or VEGFR-3 + lymphatics (right, n = 6). (C) Heart sections stained with an anti-LYVE-1 antibody (red), TRITC-labeled WGA (green) and DAPI (blue) (left, scale bar: 50 mum) and the LYVE-1 + vessel to CM ratio (right, n = 6). (D) Immunohistochemical staining of heart sections with an antibody against LYVE-1, VEGFR-3 or Podoplanin (left, scale bar: 50 mum) and quantification of LYVE-1 + , VEGFR-3 + , and Podoplanin + lymphatic vessels (right, n = 6). (E) Gravimetric assessment of cardiac water content (%) as determined by the cardiac dry weight to wet weight ( n = 6). (F) Representative color-coded images of cardiac MRI T2 mapping in which the turquoise/blue areas are normal tissues and the red/yellow areas exhibit cardiac edema (left) and MRI-based quantification of cardiac water content (T2 map signal intensity, msec) (right, n = 4). (G) Immunoblot analysis of the VEGFR-3, p-AKT, AKT, p-ERK1/2, ERK1/2, CaNA, NFATc1, FOX2C, and CX43 proteins in the heart (left) and quantification of these proteins (right, n = 4). GAPDH was used as an internal control. The data are presented as t

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Experimental details: FIGURE 2 Knockdown of VEGFR-3 impairs cardiac lymphangiogenesis and causes edema after pressure overload. VEGFR-3 knockdown (VEGFR-3 f/- ) mice and their WT (VEGFR-3 f/f ) littermates were subjected to sham or TAC surgery and remained under sham or TAC conditions for 6 weeks. (A) Heart sections stained with an antibody against LYVE-1 (red) or VEGFR-3 (green) (left, scale bar: 50 mum) and quantification of LYVE-1 + or VEGFR-3 + lymphatic vessels (right, n = 6). (B) Heart sections stained with an anti-LYVE-1 antibody (red), TRITC-labeled WGA (green) and DAPI (blue) (left, scale bar: 50 mum) and the LYVE-1 + vessel to CM ratio (right, n = 6). (C) Immunoblot analysis of the VEGFR-3, p-AKT, AKT, p-ERK1/2, ERK1/2, calcineurin A (CaNA), NFATc1, FOXC2, CX43, VEGF-D, and VEGFR-2 proteins in the heart (left) and quantification of these proteins (right, n = 4). GAPDH was used as an internal control. (D) Gravimetric assessment of the cardiac water content (%) as determined by the cardiac dry weight-to-wet weight ratio ( n = 6). The data are presented as the mean +- SD, and n represents the number of animals per group. Statistical analysis was performed with one-way ANOVA; * p < 0.05, ** p < 0.01, and *** p < 0.001 versus VEGFR-3 f/f + sham; # p < 0.05, ## p < 0.01, and ### p < 0.001 versus VEGFR-3 f/f + TAC

Supportive validation

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Experimental details: Figure 1 Metformin attenuates Il1b and Il6 mRNA induction but does not inhibit NF-kappaB activation (A) BMDM pretreated -/+ metformin (0.5 mM, 16 h) were harvested 30 min after LPS (100 ng/mL) addition. Nuclear (Nuc) and cytosolic (Cyt) fractions were analyzed for the indicated proteins. s.e., short exposure; l.e., long exposure. One representative immunoblot (IB) out of 2. (B) IB analysis of p62, DRP1, and VDAC in mitochondria from BMDM pretreated -/+ metformin, primed with LPS, and challenged with ATP (4 mM, 1 h). (C) Q-PCR quantitation of Il1b , Il6 and Il10 mRNAs before or after LPS stimulation -/+ metformin pre-treatment (n = 3). (D) p65, C/EBPbeta or NFATc1-4 recruitments to the Il1 and Il6 promoters in BMDM before or after LPS stimulation was analyzed by ChIP-qPCR assay (n = 3-7). (E) IB analysis of p38 MAPK and JNK phosphorylation (left) and quantitation of Il6 mRNA (top) and IL-6 secretion (bottom) from BMDM pretreated with metformin, SB202190 (10 muM) or SP600125 (40 muM) for 16 h and stimulated with LPS for 30 min. All IB show one representative out of 3. Results in (C-E) are averages +-SD. * p < 0.05; ** p < 0.01; *** p < 0.001. Two-sided unpaired t test. See also Figure S1. Figure S1

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Experimental details: Fig. 3 Nuclear accumulation of NFATc2 in cells treated with OPN-CMs. a Immunofluorescent images for NFATc2 subcellular localization in cells induced by OPNc-CM and CM with addition of OPN-neutralizing antibody or FK506 as an inhibitor to calcineurin. Twenty individual cells were selected for quantitative analyses of the ratio between nuclear localization and cytoplasmic localization for each NFATc2 as shown in the plot. b Quantification of NFATc1-c4 nuclear localization from immunofluorescence in cells treated with OPN-CMs for 3 h. c Western blot analyses for cytoplasm or nuclear distribution of NFATc2 and NFATc3 proteins in cells treated with OPN-CMs, where tubulin and coilin were used as cytoplasm and nuclear makers respectively. d Western blot analyses for cytoplasm or nuclear distribution of NFATc2 and NFATc3 proteins in cells treated with OPNc-CM and CM with addition of OPN-neutralizing antibody or FK506. * p < 0.05, ** p < 0.01, *** p < 0.001

Supportive validation

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Experimental details: FIGURE 1 DYRK1A and NFATC1 protein expression were coordinately increased in brain gliomas. A Representative images for the immunohistochemical staining of DYRK1A and NFATC1 in four different grades of gliomas and normal tissues. DYRK1A was detected using a polyclonal anti-DYRK1A antibody (CST, #2771) and NFATC1 was detected using a monoclonal antibody (Thermo Fisher, #MA3-024) in the microarray (Biomax, #GL481 and T173a). B. DYRK1A protein expression was increased in gliomas. The expression of DYRK1A was quantified by ImageJ software as described in the methods section. C. NFATC1 protein levels were increased in gliomas. The expression of NFATC1 was quantified by ImageJ software as described in the methods section. D. Correlation between protein levels of NFATC1 and DYRK1A in tissues was analyzed by Spearman's rank correlation test. p < 0.0001. rho = 0.0642. n =48. E. NFATC1 and DYRK1A were detected by WB in the cell lysate of NHA, U87, U251, T98G, and HEK293 cell lines. NFATC1 monoclonal antibody and DYRK1A polyclonal antibody were used as described in the methods section. F. Colocalization images for the immunofluorescent staining of DYRK1A and NFATC1 in U251 cells. DYRK1A was detected using a polyclonal anti-DYRK1A antibody (CST, #2771), and NFATC1 was detected using a monoclonal antibody (Thermo Fisher, #MA3-024), mouse IgG (Santa Cruz, sc-2025), and rabbit IgG (Proteintech, B900610) as negative controls. Values represent means +-SD, n = 3.

Supportive validation

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Experimental details: Fig. 3 Nr2f6 overexpression suppresses Th17 differentiation in vitro . (A) Wildtype ( Nr2f6 +/+ ) or Nr2f6- Tg85 (Tg85) CD4 + cells were differentiated under Th17 conditions, and Il17a , (B) Il17f , (C) Il21 , (D) Rorc , (E) and Il22 transcript levels from both genotypes were determined by qRT-PCR. The data shown are derived from three independent experiments and are normalized to the amounts of Gapdh expression. Expression in the Th17 Nr2f6 +/+ differentiated cells subset was arbitrarily set to 100 in order to facilitate comparison between the different cytokines. Error bars denote the mean +- s.e.m. * p < 0.05. The error bars denote mean +- s.e.m. * p < 0.05. (F) Expression of Nr2f6 in Nr2f6 +/+ or Nr2f6- Tg85 un-stimulated or Th17-differentiated CD4 + T cells. Note the approximate 4-fold increase in expression in the Tg85 transgenic NR2F6 cells. (G) Supernatants from wildtype ( Nr2f6 +/+ ) or Nr2f6- Tg85 (Tg85) CD4 + Th17-differentiated cells were used to measure IL-17 secretion via BioPlex. (H) EMSA analysis of nuclear extracts prepared from wildtype ( Nr2f6 +/+ ) or Tg85 CD4 + Th17-differentiated cells hybridized to the min Il17a (NFAT o3) oligonucleotide and supershifted with an NFATc1 antibody. Equal NFAT nuclear translocation was observed by western blot, and DNA polymerase delta was used as the loading control. One of three independent experiments is shown.

Supportive validation

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Experimental details: Fig. 6 NR2F6 suppresses NFAT and RORgammat binding to the CNS2 region of the Il17a promoter. (A) GST-NR2F6 binds to the CNS2 Il17a RORgammat oligonucleotide [16] of the Il17a CNS2 promoter region in EMSA analysis. (B) EMSA analysis of nuclear extracts from wildtype (wt) or Rorc -/- CD4 + T cells stimulated under Th17 conditions for 3 days and hybridized to the CNS2 Il17a ROR oligonucleotide. The specific binding of RORgammat was confirmed using a RORgammat supershift antibody loading was controlled by performing western blots with nuclear extracts probed with DNA polymerase delta no gross differences could be observed between the genotypes within the same stimulation conditions. (C) EMSA analysis of nuclear extracts of Nr2f6 +/+ or Nr2f6 -/- CD4 + T cells either left unstimulated (M) or stimulated under Th17 conditions for 3 days and hybridized to a CNS2 Il17a NFAT-specific oligonucleotide. (D) EMSA analysis of nuclear extracts of wildtype ( Nr2f6 +/+ ) or Nr2f6- Tg85 CD4 + T cells stimulated under Th17 conditions for 3 days and hybridized to a CNS2 IL17a NFAT-specific oligonucleotide. The specific binding of NFATc1 was confirmed using the NFATc1 supershift antibody and cold (c.c. oligo) and mutant competition oligonucleotides (c.m. oligo). (E) EMSA analysis of nuclear extracts from Nr2f6 +/+ or Nr2f6 -/- CD4 + T cells stimulated under Th1 or under Th17 conditions for 3 days and hybridized to the CNS2 Il17a RORgamma oligonucleotide. (F) EMSA analysis of nuclear extracts from

Supportive validation

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Experimental details: MiRNA-1224-5p inactivates the ADCY2-dependent Rap1 signaling pathway. a Gene expression levels of BMMs using agomiRNA-1224 and antagomiRNA-1224 24 h after transfection. The expression of 460 genes was upregulated (red), and the expression of 670 genes was downregulated (blue). Each group was treated with 30 ng/ml M-CSF. b Heatmap showing genes related to osteoclasts. c RT-PCR quantitatively confirmed that the expression of key genes in osteoclasts was upregulated. d KEGG pathway analysis predicts that the function of the Rap1 signaling pathway will change. e Heatmap shows genes related to the Rap1 pathway. f Western blot analysis showed that 6 h after treatment with agomiRNA-1224 in BMMs, the level of Rap1 decreased significantly. g Western blotting confirmed that treatment of BMMs with agomiRNA-1224 decreased the RANKL-induced protein expression of CDC42, p-Rac1, p-Rho-A, FAK, and Rac1 for 24 h. h Representative immunohistochemical staining showing that agomiRNA-1224 injection decreased ADCY2 and Rap1 levels in vivo. Femurs were collected within 24 h of miRNA after injection. Scale bar, 50 um. i Western blotting confirmed that treatment of BMMs with agomiRNA-1224 decreased the expression of NFATC1 protein induced by RANKL for 48 h. j ChIP experiment results show that agomiRNA-1224 induces ADCY2 to occupy the Nfatc1-binding region together. k Western blot analysis of Nfatc1. The removal of ADCY2 weakened the expression of Nfatc1 induced by agomiRNA-1224. l ChIP experiment res