MA1-025

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of NFATc2 using NFATc2 Monoclonal Antibody (25A10.D6.D2) (Product # MA1-025) shows staining in Hela Cells. NFATc2 (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 NFATc2 (Product # MA1-025) 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.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of NFATc2 using NFATc2 Monoclonal Antibody (25A10.D6.D2) (Product # MA1-025) shows staining in MCF-7 Cells. NFATc2 (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 NFATc2 (Product # MA1-025) 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.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of NFATc2 using NFATc2 Monoclonal Antibody (25A10.D6.D2) (Product # MA1-025) shows staining in U251 Cells. NFATc2 (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 NFATc2 (Product # MA1-025) 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.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of NFATC2 Monoclonal Antibody (25A10.D6.D2) was performed using 70% confluent log phase HeLa and IMR32 cells. The cells were fixed with 4% Paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 2% BSA for 10 minutes at room temperature. The cells were labeled with NFATC2 Monoclonal Antibody (Product # MA1-025) at 1:100 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A28175, 1:2000 dilution) for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b: Blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing nuclear and cytoplasmic localization. Panel e represents IMR-32 cells having no expression of NFATC2. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of NFATc2 (green) in HeLa cells. Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA (Product # 37525) for 15 minutes at room temperature. Cells were left untreated (left panel) or treated with 1uM staurosporine (right panel) for 3 hours and probed with a NFATc2 monoclonal antibody (Product # MA1-025), at a dilution of 1:100 for at least 1 hour at room temperature, washed with PBS, and incubated with DyLight 488 goat anti-mouse IgG secondary antibody (Product # 35502) at a dilution of 1:400 for 30 minutes at room temperature. F-Actin (red) was stained with DyLight 554 Phalloidin (Product # 21834) and nuclei (blue) were stained with Hoechst 33342 dye (Product # 62249). Images were taken on a Thermo Scientific ArrayScan and ToxInsight at 20X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry was performed on cancer biopsies of deparaffinized Human colon carcinoma tissues. 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 NFATc2 (Product # MA1-025) 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

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry was performed on normal deparaffinized Human spleen tissue tissues. 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 NFATc2 (Product # MA1-025) 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

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry was performed on normal deparaffinized Human tonsil tissue tissues. 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 NFATc2 (Product # MA1-025) 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

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 1 NFAT1 is expressed in HSC lines and increases following stimulation with Flt3-L Western blot analysis of un-stimulated and Flt3-L stimulated VIVIT expressing and control HSC lines. PMA treated splenic T cells were used as a positive control. qPCR analysis of NFAT1 mRNA expression in HSC lines after stimulation with Flt3-L. qPCR analysis of NFAT2 and NFAT1 expression in freshly isolated lineage (lin - ) negative cells from BM stimulated with Flt3-L. Confocal images of VIVIT-eGFP and control-tdTomato expressing HSC lines stimulated for 48 h with Flt3-L. NFAT translocation was induced by treatment with ionomycin for 2 h (original magnification, x200, scale bar 2 um). Data are representative of three ( A and C ) and five ( B ) independent experiments (mean +- SEM). Three mice per group for each time-point were used ( C ).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 5. Calcium-dependent SOCE/NFAT pathway is activated by IR in naive T-lymphocytes. (A) Distribution of endogenous Orai1 (magenta, first column) and STIM1 (green, second column) in fixed PBMCs immunostained with secondary antibodies Alx488 and Alx647, respectively. A merge of the two channels is shown in the third column, with higher magnification of marked areas in the fourth column. The merge of untreated control cells additionally shows the nucleus stained with Hoechst DNA dye (blue). Images show cells that were fixed as untreated/nonirradiated control cells (ctrl, top row) and cells fixed 15 min after treatment with 2 uM Tg (second row) or after exposing cells to 5 Gy (third row). (B) Mean ratio (+- SD, number of cells in brackets) of green fluorescence in ROI (inset image, red circle) in cytosol divided by fluorescence in ROI in direct vicinity over PM (black circle). Data from untreated control cells (ctrl) as well as cells exposed to 2 muM Tg or 5 Gy x rays 60 min after treatment. (C) Confocal images of PBMCs showing nucleus stained with Hoechst DNA dye (blue, first column) and endogenous NFATc2 (green, second column) stained with Alx488. Overlay of both columns is shown in third column. Cells were fixed immediately (untreated/nonirradiated control, top row), 15 min after 2 uM Tg Ca 2+ store depletion (second row) or 60 min after x-ray exposure with 5 Gy (third/fourth row). All scale bars, 10 mum. (D) Mean ratio (+- SD, number of cells in brackets) of GFP fluoresc

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 7. Nuclear translocation of Ca 2+ -dependent NFAT in Jurkat cells. (A) Confocal images of Jurkat cells in which nucleus was stained with Hoechst DNA dye (blue, first column) and endogenous NFATc2 immunostained with secondary antibody Alx488 (green, second column). The third column shows a merge of blue and green channels. Cells were fixed immediately in untreated/nonirradiated control cells (ctrl, first row), 15 min after 2 uM Tg Ca 2+ store depletion (Tg, second row), or 15, 30, and 60 min after x-ray exposure with 5 Gy (three bottom rows). (B) Mean ratio (+- SD, number of cells in brackets) of GFP fluorescence in nucleus (inset, magenta circle) divided by fluorescence of total cell (white circle). Statistical differences between treatments were analyzed by unpaired Student's t test, and respective P values are given in the figure. (C) Live-cell imaging of nuclear import of transiently expressed NFATc2-GFP from cytosol (c) to nucleus (n) in Jurkat cells after stimulation with 2 muM Tg in absence (top) or presence (bottom) of 10 uM CRAC channel inhibitor Synta66. Numbers indicate time in minutes after treatment. (D) Kinetic analysis of NFATc2-GFP nuclear translocation from cytosol (black) to nucleus (red). Data are from confocal imaging of Jurkat cells in untreated control condition (crtl), with 2 uM Tg in 25 mul/ml activator (T-Ac), or after 5 Gy x-ray exposure (5 Gy). Data were obtained without (left) and with (right) 10 uM Synta66. Each time course diagram is the me

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 5. GSK3beta is required for the anti-hypertrophic role of SIRT2 deacetylase. ( A ) Western blotting analysis depicting the activity of GSK3 inhibitor X (GSK3-X). Neonatal rat cardiomyocytes were treated with vehicle or 500 nM GSK3-X for 48 hr and the activity of GSK3 was assessed by monitoring the phosphorylation of GS by specific antibody. ( B ) [ 3 H]-leucine incorporation into total cellular protein of control (Ad-GFP) or SIRT2-overexpressing (Ad-SIRT2) rat neonatal cardiomyocytes treated with either vehicle or 500 nM GSK3 inhibitor X (GSK3-X) for 48 hr. Cardiomyocytes were infected with adenoviral vectors encoding either GFP or SIRT2 for 24 hr prior to GSK3-X treatment. After the GSK3-X treatment, cardiomyocytes were stimulated with either vehicle or 20 uM ISO for 24 hr and the [ 3 H]-leucine incorporation was monitored. c.p.m. counts per minute. n = 10. Data is presented as mean +- s.d. *p

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 7. Effects of LGG and Butyrate (But) on TGFb1 Production by BM T Cells, onNFAT1/2 and SMAD2/3 Activation, and on PI3K and Akt Signaling in BMCD8 + T Cells (A-C) TGFb1 mRNA expression by FACS-sorted BM conventionaleGFPCD4 + T cells, eGFPCD8 + T cells, andeGFP + Treg cells. DEREG (eGFP.Foxp3) reporter mice were treatedwith vehicle, LGG, or butyrate for 4 weeks. BM cells were sorted at the end ofthe treatment period. (D) Immunoblot analysis for the detection of NFAT1, NFAT2, pSMAD2, andpSMAD3 in purified BM CD8 + T cells. (E) Immunoblot analysis for the detection of c-Jun and c-Fos inpurified BM CD8 + T cells. (D and E) Fresh BM CD8 + T cells were pooled together andthen used for obtaining nuclear and cytoplasmic fractions. Laminin B1 was usedas nuclear loading control. Tubulin was used as cytoplasmic loading control. (F) Immunoblot analysis for the detection of Phospho-PI3K p85 in thewhole lysate from BM CD8 + T cells. (G and H) pAKT levels in BM CD8 + T cells and percent ofpAKT + BM CD8 + T cells, as determined by flowcytometry. (D-F) Conventionally raised WT mice were treated with vehicle,LGG, or butyrate for 4 weeks. BM CD8 + T cells were purified at theend of the treatment period using the EasySep Mouse CD8 + T CellIsolation Kit. One representative experiment of 3 experiments. Data were expressed as mean +- SEM, n = 5-10 mice pergroup in (A)-(C), (G), and (H). Data were analyzed by two-way ANOVA andpost hoc tests applying the Bonferroni correction for multiple compar