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Q-RT-PCR validation of gene expression changes observed following microarray analysis of <t>PBMCs</t> from control and infected cows. Genes to be validated were selected from a list of the genes whose expression was most significantly different following nil stimulation of PBMCs from infected and control cows. Q-RT-PCR was performed as described in Materials and Methods, and data were analyzed by using the 2 −ΔΔCt method with β-actin as the control gene. Mean values for control cow PBMCs with nil stimulation or M. <t>avium</t> subsp. paratuberculosis (MPTb) stimulation were used as calibrators for calculation of all 2 −ΔΔCt values, so that the values for control cows always bracketed 1.0. For Q-RT-PCR analysis, samples were arranged in 96-well PCR plates so that comparisons could be made between PBMCs from infected cows and PBMCs from control cows, each stimulated with M. avium subsp. paratuberculosis or PBS (nil stimulation), on the same plate. The data are means ± standard errors of the means for 2 −ΔΔCt values for three or four infected cows and three or four control cows for each gene.
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Images

1) Product Images from "Evidence for a Novel Gene Expression Program in Peripheral Blood Mononuclear Cells from Mycobacterium avium subsp. paratuberculosis-Infected Cattle "

Article Title: Evidence for a Novel Gene Expression Program in Peripheral Blood Mononuclear Cells from Mycobacterium avium subsp. paratuberculosis-Infected Cattle

Journal: Infection and Immunity

doi: 10.1128/IAI.71.11.6487-6498.2003

Q-RT-PCR validation of gene expression changes observed following microarray analysis of PBMCs from control and infected cows. Genes to be validated were selected from a list of the genes whose expression was most significantly different following nil stimulation of PBMCs from infected and control cows. Q-RT-PCR was performed as described in Materials and Methods, and data were analyzed by using the 2 −ΔΔCt method with β-actin as the control gene. Mean values for control cow PBMCs with nil stimulation or M. avium subsp. paratuberculosis (MPTb) stimulation were used as calibrators for calculation of all 2 −ΔΔCt values, so that the values for control cows always bracketed 1.0. For Q-RT-PCR analysis, samples were arranged in 96-well PCR plates so that comparisons could be made between PBMCs from infected cows and PBMCs from control cows, each stimulated with M. avium subsp. paratuberculosis or PBS (nil stimulation), on the same plate. The data are means ± standard errors of the means for 2 −ΔΔCt values for three or four infected cows and three or four control cows for each gene.
Figure Legend Snippet: Q-RT-PCR validation of gene expression changes observed following microarray analysis of PBMCs from control and infected cows. Genes to be validated were selected from a list of the genes whose expression was most significantly different following nil stimulation of PBMCs from infected and control cows. Q-RT-PCR was performed as described in Materials and Methods, and data were analyzed by using the 2 −ΔΔCt method with β-actin as the control gene. Mean values for control cow PBMCs with nil stimulation or M. avium subsp. paratuberculosis (MPTb) stimulation were used as calibrators for calculation of all 2 −ΔΔCt values, so that the values for control cows always bracketed 1.0. For Q-RT-PCR analysis, samples were arranged in 96-well PCR plates so that comparisons could be made between PBMCs from infected cows and PBMCs from control cows, each stimulated with M. avium subsp. paratuberculosis or PBS (nil stimulation), on the same plate. The data are means ± standard errors of the means for 2 −ΔΔCt values for three or four infected cows and three or four control cows for each gene.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Microarray, Infection, Polymerase Chain Reaction

Q-RT-PCR validation of cDNA microarray results for PBMCs from control cows. Genes encoding Sentrin-(SUMO-1), MMP 1, and MMP 23 were selected for Q-RT-PCR validation from among the genes that exhibit differential expression in nil-stimulated and M. avium subsp. paratuberculosis -stimulated PBMCs from control uninfected cows. The gene encoding Sentrin-(SUMO-1) was selected as a representative of genes that exhibit up-regulation in control cow PBMCs stimulated with M. avium subsp. paratuberculosis compared to expression in nil-stimulated cells. The genes encoding MMP 1 and MMP 23 were selected because each was apparently down-regulated by M. avium subsp. paratuberculosis stimulation of control cow PBMCs on cDNA microarrays and because of previous data suggesting that MMP gene regulation is a major and consistent effect of M. avium subsp. paratuberculosis ). For these reasons, an analysis of nil-stimulated and M. avium subsp. paratuberculosis -stimulated PBMCs from infected cows was also included in this study. Q-RT-PCR was conducted as described in Materials and Methods by using gene-specific primers. Data were analyzed by using the 2 −ΔΔCt ) with β-actin as the control gene and nil stimulation within animal as the calibrator. The data are the means ± standard errors of the means for independent results from four infected cows and three control cows. MPTb, M. avium subsp. paratuberculosis .
Figure Legend Snippet: Q-RT-PCR validation of cDNA microarray results for PBMCs from control cows. Genes encoding Sentrin-(SUMO-1), MMP 1, and MMP 23 were selected for Q-RT-PCR validation from among the genes that exhibit differential expression in nil-stimulated and M. avium subsp. paratuberculosis -stimulated PBMCs from control uninfected cows. The gene encoding Sentrin-(SUMO-1) was selected as a representative of genes that exhibit up-regulation in control cow PBMCs stimulated with M. avium subsp. paratuberculosis compared to expression in nil-stimulated cells. The genes encoding MMP 1 and MMP 23 were selected because each was apparently down-regulated by M. avium subsp. paratuberculosis stimulation of control cow PBMCs on cDNA microarrays and because of previous data suggesting that MMP gene regulation is a major and consistent effect of M. avium subsp. paratuberculosis ). For these reasons, an analysis of nil-stimulated and M. avium subsp. paratuberculosis -stimulated PBMCs from infected cows was also included in this study. Q-RT-PCR was conducted as described in Materials and Methods by using gene-specific primers. Data were analyzed by using the 2 −ΔΔCt ) with β-actin as the control gene and nil stimulation within animal as the calibrator. The data are the means ± standard errors of the means for independent results from four infected cows and three control cows. MPTb, M. avium subsp. paratuberculosis .

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Microarray, Expressing, Infection

Identification of numerous gene expression changes in both nil-stimulated and M. avium subsp. paratuberculosis -stimulated PBMCs when expression levels were compared across infection groups by using a mixed-model analysis. Data from microarray analysis of PBMCs from six infected cows and four control cows were combined and analyzed as described in Materials and Methods by using a two-stage mixed model in SAS. The resulting least square (LS) means were used to construct interaction tables containing relative expression information and confidence intervals for each gene on the BOTL-3 cDNA microarray. Data were imported into Excel, and the Data Filter command was used to select genes with various expression differences (fold changes) and significance values ( P
Figure Legend Snippet: Identification of numerous gene expression changes in both nil-stimulated and M. avium subsp. paratuberculosis -stimulated PBMCs when expression levels were compared across infection groups by using a mixed-model analysis. Data from microarray analysis of PBMCs from six infected cows and four control cows were combined and analyzed as described in Materials and Methods by using a two-stage mixed model in SAS. The resulting least square (LS) means were used to construct interaction tables containing relative expression information and confidence intervals for each gene on the BOTL-3 cDNA microarray. Data were imported into Excel, and the Data Filter command was used to select genes with various expression differences (fold changes) and significance values ( P

Techniques Used: Expressing, Infection, Microarray, Construct

2) Product Images from "Induction of the CTLA-4 Gene in Human Lymphocytes Is Dependent on NFAT Binding the Proximal Promoter"

Article Title: Induction of the CTLA-4 Gene in Human Lymphocytes Is Dependent on NFAT Binding the Proximal Promoter

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi:

C(−280)NFAT-binding activity does not depend on AP-1 cooperativity and has a higher affinity than NFAT sites with AP-1 cooperativity. a, NFAT binding to C(−280)NFAT is independent of AP-1. AP-1 sequence failed to compete for binding by C(−280)NFAT in EMSA using human PBMC extracts and vice versa. C(−280)NFAT failed to compete against AP-1 binding. *C-NFAT probe, C(−280)NFAT; *AP-1 probe, AP-1 oligonucleotide. b, NFAT-binding complex does not contain AP-1 components. Abs to Jun-B or c-Fos when incubated with PBMC extracts in EMSAs do not alter binding to the C(−280)NFAT probe. c, NFAT DNA-binding kinetic analysis. NFAT binding to C(−280)NFAT is more stable than to NFAT:AP-1 sites from the human and murine IL-2 promoter in PBMC extracts. Excess competitor for the indicated sites was added and the duration of binding was measured by EMSA. Right panel, The absorbance (OD) of the binding complex after addition of excess competitor. d, NFAT promoter sequence comparison. C(−280)NFAT sequence has four adenosine bases (shadowed) in common when aligned with NFAT IL-2 promoter sequences. Underlined adenosine bases are critical for DNA binding as determined by EMSA ( e ). Sp = species; h = human; m = mouse; D = A,T; N = A,C,T,G; X = A,T,C; a = noncoding sequence; b = EMSA competitor. e, Mutation analysis of C(−280)NFAT to identify nucleotides important for DNA binding. Single-base pair substitutions (C↔A and G↔T) across the C(−280)NFAT sequence were introduced into the indicated nucleotide positions and the corresponding double-stranded oligonucleotides containing mutations were used as competitor sequence in EMSA.
Figure Legend Snippet: C(−280)NFAT-binding activity does not depend on AP-1 cooperativity and has a higher affinity than NFAT sites with AP-1 cooperativity. a, NFAT binding to C(−280)NFAT is independent of AP-1. AP-1 sequence failed to compete for binding by C(−280)NFAT in EMSA using human PBMC extracts and vice versa. C(−280)NFAT failed to compete against AP-1 binding. *C-NFAT probe, C(−280)NFAT; *AP-1 probe, AP-1 oligonucleotide. b, NFAT-binding complex does not contain AP-1 components. Abs to Jun-B or c-Fos when incubated with PBMC extracts in EMSAs do not alter binding to the C(−280)NFAT probe. c, NFAT DNA-binding kinetic analysis. NFAT binding to C(−280)NFAT is more stable than to NFAT:AP-1 sites from the human and murine IL-2 promoter in PBMC extracts. Excess competitor for the indicated sites was added and the duration of binding was measured by EMSA. Right panel, The absorbance (OD) of the binding complex after addition of excess competitor. d, NFAT promoter sequence comparison. C(−280)NFAT sequence has four adenosine bases (shadowed) in common when aligned with NFAT IL-2 promoter sequences. Underlined adenosine bases are critical for DNA binding as determined by EMSA ( e ). Sp = species; h = human; m = mouse; D = A,T; N = A,C,T,G; X = A,T,C; a = noncoding sequence; b = EMSA competitor. e, Mutation analysis of C(−280)NFAT to identify nucleotides important for DNA binding. Single-base pair substitutions (C↔A and G↔T) across the C(−280)NFAT sequence were introduced into the indicated nucleotide positions and the corresponding double-stranded oligonucleotides containing mutations were used as competitor sequence in EMSA.

Techniques Used: Binding Assay, Activity Assay, Sequencing, Incubation, Mutagenesis

Identifying NFAT as the factor that binds to the proximal CTLA-4 promoter. a, Sequence of the proximal CTLA-4 promoter important for transcription and potential transcription factor recognition motifs predicted by Transcription ESS is shown from nucleotides −264 to −297 from the translational start site. This sequence has consensus sites for NFAT, c-Myb, and SRE and is defined as C(−280)NFAT. b, NFAT sequences compete away DNA binding to C(−280)NFAT probe. EMSA results with γ - 32 P-labeled dsC(−280)NFAT probe, with extracts prepared from stimulated human PBMCs. Competition assays were performed using excess double-stranded unlabeled oligonucleotides for C(−280)NFAT = C-NFAT, human IL-2 NFAT = hNFAT, Sp-1, or GAS sites. c, C(−280)NFAT oligonucleotide interacts with DNA-binding activity for NFAT and is competed by other NFAT sequences (hIL-2 NFAT and mIL-2 NFAT). Competition with excess c-Myb or SRE oligonucleotide does not alter DNA binding to the C(−280)NFAT probe. d, NFAT1 binds to C(−280)NFAT. EMSA performed in the presence of Abs specific to NFAT1 yields a supershifted band (arrow) whereas nonspecific Ab (Ns Ab) or anti-NFAT2 does not.
Figure Legend Snippet: Identifying NFAT as the factor that binds to the proximal CTLA-4 promoter. a, Sequence of the proximal CTLA-4 promoter important for transcription and potential transcription factor recognition motifs predicted by Transcription ESS is shown from nucleotides −264 to −297 from the translational start site. This sequence has consensus sites for NFAT, c-Myb, and SRE and is defined as C(−280)NFAT. b, NFAT sequences compete away DNA binding to C(−280)NFAT probe. EMSA results with γ - 32 P-labeled dsC(−280)NFAT probe, with extracts prepared from stimulated human PBMCs. Competition assays were performed using excess double-stranded unlabeled oligonucleotides for C(−280)NFAT = C-NFAT, human IL-2 NFAT = hNFAT, Sp-1, or GAS sites. c, C(−280)NFAT oligonucleotide interacts with DNA-binding activity for NFAT and is competed by other NFAT sequences (hIL-2 NFAT and mIL-2 NFAT). Competition with excess c-Myb or SRE oligonucleotide does not alter DNA binding to the C(−280)NFAT probe. d, NFAT1 binds to C(−280)NFAT. EMSA performed in the presence of Abs specific to NFAT1 yields a supershifted band (arrow) whereas nonspecific Ab (Ns Ab) or anti-NFAT2 does not.

Techniques Used: Sequencing, Binding Assay, Labeling, Activity Assay

Identifying NFAT as the factor that binds to the proximal CTLA-4 promoter. a, Sequence of the proximal CTLA-4 promoter important for transcription and potential transcription factor recognition motifs predicted by Transcription ESS is shown from nucleotides −264 to −297 from the translational start site. This sequence has consensus sites for NFAT, c-Myb, and SRE and is defined as C(−280)NFAT. b, NFAT sequences compete away DNA binding to C(−280)NFAT probe. EMSA results with γ - 32 P-labeled dsC(−280)NFAT probe, with extracts prepared from stimulated human PBMCs. Competition assays were performed using excess double-stranded unlabeled oligonucleotides for C(−280)NFAT = C-NFAT, human IL-2 NFAT = hNFAT, Sp-1, or GAS sites. c, C(−280)NFAT oligonucleotide interacts with DNA-binding activity for NFAT and is competed by other NFAT sequences (hIL-2 NFAT and mIL-2 NFAT). Competition with excess c-Myb or SRE oligonucleotide does not alter DNA binding to the C(−280)NFAT probe. d, NFAT1 binds to C(−280)NFAT. EMSA performed in the presence of Abs specific to NFAT1 yields a supershifted band (arrow) whereas nonspecific Ab (Ns Ab) or anti-NFAT2 does not.
Figure Legend Snippet: Identifying NFAT as the factor that binds to the proximal CTLA-4 promoter. a, Sequence of the proximal CTLA-4 promoter important for transcription and potential transcription factor recognition motifs predicted by Transcription ESS is shown from nucleotides −264 to −297 from the translational start site. This sequence has consensus sites for NFAT, c-Myb, and SRE and is defined as C(−280)NFAT. b, NFAT sequences compete away DNA binding to C(−280)NFAT probe. EMSA results with γ - 32 P-labeled dsC(−280)NFAT probe, with extracts prepared from stimulated human PBMCs. Competition assays were performed using excess double-stranded unlabeled oligonucleotides for C(−280)NFAT = C-NFAT, human IL-2 NFAT = hNFAT, Sp-1, or GAS sites. c, C(−280)NFAT oligonucleotide interacts with DNA-binding activity for NFAT and is competed by other NFAT sequences (hIL-2 NFAT and mIL-2 NFAT). Competition with excess c-Myb or SRE oligonucleotide does not alter DNA binding to the C(−280)NFAT probe. d, NFAT1 binds to C(−280)NFAT. EMSA performed in the presence of Abs specific to NFAT1 yields a supershifted band (arrow) whereas nonspecific Ab (Ns Ab) or anti-NFAT2 does not.

Techniques Used: Sequencing, Binding Assay, Labeling, Activity Assay

3) Product Images from "Induction of the CTLA-4 Gene in Human Lymphocytes Is Dependent on NFAT Binding the Proximal Promoter 1"

Article Title: Induction of the CTLA-4 Gene in Human Lymphocytes Is Dependent on NFAT Binding the Proximal Promoter 1

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi:

C(−280)NFAT-binding activity does not depend on AP-1 cooperativity and has a higher affinity than NFAT sites with AP-1 cooperativity. a, NFAT binding to C(−280)NFAT is independent of AP-1. AP-1 sequence failed to compete for binding by C(−280)NFAT in EMSA using human PBMC extracts and vice versa. C(−280)NFAT failed to compete against AP-1 binding. *C-NFAT probe, C(−280)NFAT; *AP-1 probe, AP-1 oligonucleotide. b, NFAT-binding complex does not contain AP-1 components. Abs to Jun-B or c-Fos when incubated with PBMC extracts in EMSAs do not alter binding to the C(−280)NFAT probe. c, NFAT DNA-binding kinetic analysis. NFAT binding to C(−280)NFAT is more stable than to NFAT:AP-1 sites from the human and murine IL-2 promoter in PBMC extracts. Excess competitor for the indicated sites was added and the duration of binding was measured by EMSA. Right panel, The absorbance (OD) of the binding complex after addition of excess competitor. d, NFAT promoter sequence comparison. C(−280)NFAT sequence has four adenosine bases (shadowed) in common when aligned with NFAT IL-2 promoter sequences. Underlined adenosine bases are critical for DNA binding as determined by EMSA ( e ). Sp = species; h = human; m = mouse; D = A,T; N = A,C,T,G; X = A,T,C; a = noncoding sequence; b = EMSA competitor. e, Mutation analysis of C(−280)NFAT to identify nucleotides important for DNA binding. Single-base pair substitutions (C↔A and G↔T) across the C(−280)NFAT sequence were introduced into the indicated nucleotide positions and the corresponding double-stranded oligonucleotides containing mutations were used as competitor sequence in EMSA.
Figure Legend Snippet: C(−280)NFAT-binding activity does not depend on AP-1 cooperativity and has a higher affinity than NFAT sites with AP-1 cooperativity. a, NFAT binding to C(−280)NFAT is independent of AP-1. AP-1 sequence failed to compete for binding by C(−280)NFAT in EMSA using human PBMC extracts and vice versa. C(−280)NFAT failed to compete against AP-1 binding. *C-NFAT probe, C(−280)NFAT; *AP-1 probe, AP-1 oligonucleotide. b, NFAT-binding complex does not contain AP-1 components. Abs to Jun-B or c-Fos when incubated with PBMC extracts in EMSAs do not alter binding to the C(−280)NFAT probe. c, NFAT DNA-binding kinetic analysis. NFAT binding to C(−280)NFAT is more stable than to NFAT:AP-1 sites from the human and murine IL-2 promoter in PBMC extracts. Excess competitor for the indicated sites was added and the duration of binding was measured by EMSA. Right panel, The absorbance (OD) of the binding complex after addition of excess competitor. d, NFAT promoter sequence comparison. C(−280)NFAT sequence has four adenosine bases (shadowed) in common when aligned with NFAT IL-2 promoter sequences. Underlined adenosine bases are critical for DNA binding as determined by EMSA ( e ). Sp = species; h = human; m = mouse; D = A,T; N = A,C,T,G; X = A,T,C; a = noncoding sequence; b = EMSA competitor. e, Mutation analysis of C(−280)NFAT to identify nucleotides important for DNA binding. Single-base pair substitutions (C↔A and G↔T) across the C(−280)NFAT sequence were introduced into the indicated nucleotide positions and the corresponding double-stranded oligonucleotides containing mutations were used as competitor sequence in EMSA.

Techniques Used: Binding Assay, Activity Assay, Sequencing, Incubation, Mutagenesis

Identifying NFAT as the factor that binds to the proximal CTLA-4 promoter. a, Sequence of the proximal CTLA-4 promoter important for transcription and potential transcription factor recognition motifs predicted by Transcription ESS is shown from nucleotides −264 to −297 from the translational start site. This sequence has consensus sites for NFAT, c-Myb, and SRE and is defined as C(−280)NFAT. b, NFAT sequences compete away DNA binding to C(−280)NFAT probe. EMSA results with γ - 32 P-labeled dsC(−280)NFAT probe, with extracts prepared from stimulated human PBMCs. Competition assays were performed using excess double-stranded unlabeled oligonucleotides for C(−280)NFAT = C-NFAT, human IL-2 NFAT = hNFAT, Sp-1, or GAS sites. c, C(−280)NFAT oligonucleotide interacts with DNA-binding activity for NFAT and is competed by other NFAT sequences (hIL-2 NFAT and mIL-2 NFAT). Competition with excess c-Myb or SRE oligonucleotide does not alter DNA binding to the C(−280)NFAT probe. d, NFAT1 binds to C(−280)NFAT. EMSA performed in the presence of Abs specific to NFAT1 yields a supershifted band (arrow) whereas nonspecific Ab (Ns Ab) or anti-NFAT2 does not.
Figure Legend Snippet: Identifying NFAT as the factor that binds to the proximal CTLA-4 promoter. a, Sequence of the proximal CTLA-4 promoter important for transcription and potential transcription factor recognition motifs predicted by Transcription ESS is shown from nucleotides −264 to −297 from the translational start site. This sequence has consensus sites for NFAT, c-Myb, and SRE and is defined as C(−280)NFAT. b, NFAT sequences compete away DNA binding to C(−280)NFAT probe. EMSA results with γ - 32 P-labeled dsC(−280)NFAT probe, with extracts prepared from stimulated human PBMCs. Competition assays were performed using excess double-stranded unlabeled oligonucleotides for C(−280)NFAT = C-NFAT, human IL-2 NFAT = hNFAT, Sp-1, or GAS sites. c, C(−280)NFAT oligonucleotide interacts with DNA-binding activity for NFAT and is competed by other NFAT sequences (hIL-2 NFAT and mIL-2 NFAT). Competition with excess c-Myb or SRE oligonucleotide does not alter DNA binding to the C(−280)NFAT probe. d, NFAT1 binds to C(−280)NFAT. EMSA performed in the presence of Abs specific to NFAT1 yields a supershifted band (arrow) whereas nonspecific Ab (Ns Ab) or anti-NFAT2 does not.

Techniques Used: Sequencing, Binding Assay, Labeling, Activity Assay

Identifying NFAT as the factor that binds to the proximal CTLA-4 promoter. a, Sequence of the proximal CTLA-4 promoter important for transcription and potential transcription factor recognition motifs predicted by Transcription ESS is shown from nucleotides −264 to −297 from the translational start site. This sequence has consensus sites for NFAT, c-Myb, and SRE and is defined as C(−280)NFAT. b, NFAT sequences compete away DNA binding to C(−280)NFAT probe. EMSA results with γ - 32 P-labeled dsC(−280)NFAT probe, with extracts prepared from stimulated human PBMCs. Competition assays were performed using excess double-stranded unlabeled oligonucleotides for C(−280)NFAT = C-NFAT, human IL-2 NFAT = hNFAT, Sp-1, or GAS sites. c, C(−280)NFAT oligonucleotide interacts with DNA-binding activity for NFAT and is competed by other NFAT sequences (hIL-2 NFAT and mIL-2 NFAT). Competition with excess c-Myb or SRE oligonucleotide does not alter DNA binding to the C(−280)NFAT probe. d, NFAT1 binds to C(−280)NFAT. EMSA performed in the presence of Abs specific to NFAT1 yields a supershifted band (arrow) whereas nonspecific Ab (Ns Ab) or anti-NFAT2 does not.
Figure Legend Snippet: Identifying NFAT as the factor that binds to the proximal CTLA-4 promoter. a, Sequence of the proximal CTLA-4 promoter important for transcription and potential transcription factor recognition motifs predicted by Transcription ESS is shown from nucleotides −264 to −297 from the translational start site. This sequence has consensus sites for NFAT, c-Myb, and SRE and is defined as C(−280)NFAT. b, NFAT sequences compete away DNA binding to C(−280)NFAT probe. EMSA results with γ - 32 P-labeled dsC(−280)NFAT probe, with extracts prepared from stimulated human PBMCs. Competition assays were performed using excess double-stranded unlabeled oligonucleotides for C(−280)NFAT = C-NFAT, human IL-2 NFAT = hNFAT, Sp-1, or GAS sites. c, C(−280)NFAT oligonucleotide interacts with DNA-binding activity for NFAT and is competed by other NFAT sequences (hIL-2 NFAT and mIL-2 NFAT). Competition with excess c-Myb or SRE oligonucleotide does not alter DNA binding to the C(−280)NFAT probe. d, NFAT1 binds to C(−280)NFAT. EMSA performed in the presence of Abs specific to NFAT1 yields a supershifted band (arrow) whereas nonspecific Ab (Ns Ab) or anti-NFAT2 does not.

Techniques Used: Sequencing, Binding Assay, Labeling, Activity Assay

Related Articles

Fluorescence:

Article Title: The retinoic acid receptor-? modulators ATRA and Ro415253 reciprocally regulate human IL-5+ Th2 cell proliferation and cytokine expression
Article Snippet: .. Abbreviations RA: Retinoic acid; ATRA: All-trans retinoic acid; RARα: Retinoic acid receptor alpha; RXR: Retinoid X receptor; PBMC: Peripheral blood mononuclear cells; HDM: House dust mite; Ag: Antigen; APC: Antigen presenting cells; TCR: T cell receptor; CTV: Cell trace violet; Th2: T helper 2; Ro41: Ro41-5253; RARE: Retinoic acid response element; IL5p: Interleukin 5 gene promoter; IL13p: Interleukin 13 gene promoter; IL4p: Interleukin 4 gene promoter; mRNA: Messenger ribonucleic acid; ICCS: Intracellular cytokine staining; qRT-PCR: Quantitative real time PCR; DMSO: Dimethyl sulfoxide; DMEM: Dulbecco modified eagles medium; FBS: Fetal bovine serum; ANOVA: Analysis of variation; MFI: Mean fluorescence intensity; SEM: Standard error of mean ..

Infection:

Article Title: Evidence for a Novel Gene Expression Program in Peripheral Blood Mononuclear Cells from Mycobacterium avium subsp. paratuberculosis-Infected Cattle
Article Snippet: .. However, stimulation of PBMCs with M. avium subsp. paratuberculosis caused a marked reduction in ALG-4 gene expression in infected cow PBMCs, resulting in a mean level of expression that was twofold lower than that in similarly treated control cow PBMCs (Fig. ). .. Thus, the overall effect of M. avium subsp. paratuberculosis on ALG-4 gene expression in infected cow PBMCs compared to expression in nil-stimulated cells from the same cows was a 16-fold reduction in expression, as measured by Q-RT-PCR.

Real-time Polymerase Chain Reaction:

Article Title: The retinoic acid receptor-? modulators ATRA and Ro415253 reciprocally regulate human IL-5+ Th2 cell proliferation and cytokine expression
Article Snippet: .. Abbreviations RA: Retinoic acid; ATRA: All-trans retinoic acid; RARα: Retinoic acid receptor alpha; RXR: Retinoid X receptor; PBMC: Peripheral blood mononuclear cells; HDM: House dust mite; Ag: Antigen; APC: Antigen presenting cells; TCR: T cell receptor; CTV: Cell trace violet; Th2: T helper 2; Ro41: Ro41-5253; RARE: Retinoic acid response element; IL5p: Interleukin 5 gene promoter; IL13p: Interleukin 13 gene promoter; IL4p: Interleukin 4 gene promoter; mRNA: Messenger ribonucleic acid; ICCS: Intracellular cytokine staining; qRT-PCR: Quantitative real time PCR; DMSO: Dimethyl sulfoxide; DMEM: Dulbecco modified eagles medium; FBS: Fetal bovine serum; ANOVA: Analysis of variation; MFI: Mean fluorescence intensity; SEM: Standard error of mean ..

Activity Assay:

Article Title: Induction of the CTLA-4 Gene in Human Lymphocytes Is Dependent on NFAT Binding the Proximal Promoter
Article Snippet: .. To study the dependence of CTLA-4 expression on NFAT activity in PBMCs, cell-permeable inhibitors that affect NFAT specifically were added to PBMCs and CTLA-4 gene expression was measured. ..

Quantitative RT-PCR:

Article Title: The retinoic acid receptor-? modulators ATRA and Ro415253 reciprocally regulate human IL-5+ Th2 cell proliferation and cytokine expression
Article Snippet: .. Abbreviations RA: Retinoic acid; ATRA: All-trans retinoic acid; RARα: Retinoic acid receptor alpha; RXR: Retinoid X receptor; PBMC: Peripheral blood mononuclear cells; HDM: House dust mite; Ag: Antigen; APC: Antigen presenting cells; TCR: T cell receptor; CTV: Cell trace violet; Th2: T helper 2; Ro41: Ro41-5253; RARE: Retinoic acid response element; IL5p: Interleukin 5 gene promoter; IL13p: Interleukin 13 gene promoter; IL4p: Interleukin 4 gene promoter; mRNA: Messenger ribonucleic acid; ICCS: Intracellular cytokine staining; qRT-PCR: Quantitative real time PCR; DMSO: Dimethyl sulfoxide; DMEM: Dulbecco modified eagles medium; FBS: Fetal bovine serum; ANOVA: Analysis of variation; MFI: Mean fluorescence intensity; SEM: Standard error of mean ..

Expressing:

Article Title: Evidence for a Novel Gene Expression Program in Peripheral Blood Mononuclear Cells from Mycobacterium avium subsp. paratuberculosis-Infected Cattle
Article Snippet: .. However, stimulation of PBMCs with M. avium subsp. paratuberculosis caused a marked reduction in ALG-4 gene expression in infected cow PBMCs, resulting in a mean level of expression that was twofold lower than that in similarly treated control cow PBMCs (Fig. ). .. Thus, the overall effect of M. avium subsp. paratuberculosis on ALG-4 gene expression in infected cow PBMCs compared to expression in nil-stimulated cells from the same cows was a 16-fold reduction in expression, as measured by Q-RT-PCR.

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Article Title: Induction of the CTLA-4 Gene in Human Lymphocytes Is Dependent on NFAT Binding the Proximal Promoter
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Article Title: Dysregulated Expression of CD28 and CTLA-4 Molecules in Patients with Acute Myeloid Leukemia and Possible Association with Development of Graft versus Host Disease after Hematopoietic Stem Cell Transplantation
Article Snippet: .. It should be noted that in addition to the cells of the immune system, peripheral blood mononuclear cells also constitute a fraction of leukemic blasts, and such changes in CD28 and CTLA-4 gene expression may also be attributed to AML blasts. .. Therefore, it is better to evaluate the change in these co-stimulatory molecules and also other molecules such as ICOS and PD-1 in an isolated population of T cells as well as blasts (both peripheral blood and bone marrow-derived) from AML patients.

Article Title: The Effects of Isoproterenol and Propranolol on Cytokine Profile Secretion by Cultured Tumor-infiltrating Lymphocytes Derived from Colorectal Cancer Patients
Article Snippet: .. These results were consistent with a previous study in which IFN-γ gene expression was 94.1% in colorectal tumor tissue, 84.2% in patients' PBMCs and 40% in controls' PBMCs, with no significant differences noted in IL-4 gene expression ( ). ..

Article Title: Phytochemical and Pharmacological Properties of Capparis spinosa as a Medicinal Plant
Article Snippet: .. The authors also found that PBMC treated with the aqueous fraction of C. spinosa leaf extract had a significant increase in interleukin (IL)-4 gene expression (an anti-inflammatory cytokine) and a significant decrease in IL-17 gene expression (pro-inflammatory cytokine) [ ]. .. Therefore, these studies suggested that C. spinosa leaf extracts exhibit anti-inflammatory activity by inhibiting the pro-inflammatory cytokines expression and immune cell infiltration [ , ].

Modification:

Article Title: The retinoic acid receptor-? modulators ATRA and Ro415253 reciprocally regulate human IL-5+ Th2 cell proliferation and cytokine expression
Article Snippet: .. Abbreviations RA: Retinoic acid; ATRA: All-trans retinoic acid; RARα: Retinoic acid receptor alpha; RXR: Retinoid X receptor; PBMC: Peripheral blood mononuclear cells; HDM: House dust mite; Ag: Antigen; APC: Antigen presenting cells; TCR: T cell receptor; CTV: Cell trace violet; Th2: T helper 2; Ro41: Ro41-5253; RARE: Retinoic acid response element; IL5p: Interleukin 5 gene promoter; IL13p: Interleukin 13 gene promoter; IL4p: Interleukin 4 gene promoter; mRNA: Messenger ribonucleic acid; ICCS: Intracellular cytokine staining; qRT-PCR: Quantitative real time PCR; DMSO: Dimethyl sulfoxide; DMEM: Dulbecco modified eagles medium; FBS: Fetal bovine serum; ANOVA: Analysis of variation; MFI: Mean fluorescence intensity; SEM: Standard error of mean ..

Staining:

Article Title: The retinoic acid receptor-? modulators ATRA and Ro415253 reciprocally regulate human IL-5+ Th2 cell proliferation and cytokine expression
Article Snippet: .. Abbreviations RA: Retinoic acid; ATRA: All-trans retinoic acid; RARα: Retinoic acid receptor alpha; RXR: Retinoid X receptor; PBMC: Peripheral blood mononuclear cells; HDM: House dust mite; Ag: Antigen; APC: Antigen presenting cells; TCR: T cell receptor; CTV: Cell trace violet; Th2: T helper 2; Ro41: Ro41-5253; RARE: Retinoic acid response element; IL5p: Interleukin 5 gene promoter; IL13p: Interleukin 13 gene promoter; IL4p: Interleukin 4 gene promoter; mRNA: Messenger ribonucleic acid; ICCS: Intracellular cytokine staining; qRT-PCR: Quantitative real time PCR; DMSO: Dimethyl sulfoxide; DMEM: Dulbecco modified eagles medium; FBS: Fetal bovine serum; ANOVA: Analysis of variation; MFI: Mean fluorescence intensity; SEM: Standard error of mean ..

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    4Gene pbmcs
    Q-RT-PCR validation of gene expression changes observed following microarray analysis of <t>PBMCs</t> from control and infected cows. Genes to be validated were selected from a list of the genes whose expression was most significantly different following nil stimulation of PBMCs from infected and control cows. Q-RT-PCR was performed as described in Materials and Methods, and data were analyzed by using the 2 −ΔΔCt method with β-actin as the control gene. Mean values for control cow PBMCs with nil stimulation or M. <t>avium</t> subsp. paratuberculosis (MPTb) stimulation were used as calibrators for calculation of all 2 −ΔΔCt values, so that the values for control cows always bracketed 1.0. For Q-RT-PCR analysis, samples were arranged in 96-well PCR plates so that comparisons could be made between PBMCs from infected cows and PBMCs from control cows, each stimulated with M. avium subsp. paratuberculosis or PBS (nil stimulation), on the same plate. The data are means ± standard errors of the means for 2 −ΔΔCt values for three or four infected cows and three or four control cows for each gene.
    Pbmcs, supplied by 4Gene, used in various techniques. Bioz Stars score: 92/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    4Gene foal pbmc
    Total percentages of (A) <t>IFN-γ,</t> (B) IL-10 and (C) IL-4 producing lymphocytes in neonates ( n = 18), foals until 12 weeks of age ( n = 15) and in adult horses ( n = 15). <t>PBMC</t> were stimulated with PMA and ionomycin. The cells were stained for intracellular cytokine production and were analyzed by flow cytometry. The percentages include all cells producing the respective cytokine including double positive cells. The horizontal lines within the data sets represent medians. *** p
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    Q-RT-PCR validation of gene expression changes observed following microarray analysis of PBMCs from control and infected cows. Genes to be validated were selected from a list of the genes whose expression was most significantly different following nil stimulation of PBMCs from infected and control cows. Q-RT-PCR was performed as described in Materials and Methods, and data were analyzed by using the 2 −ΔΔCt method with β-actin as the control gene. Mean values for control cow PBMCs with nil stimulation or M. avium subsp. paratuberculosis (MPTb) stimulation were used as calibrators for calculation of all 2 −ΔΔCt values, so that the values for control cows always bracketed 1.0. For Q-RT-PCR analysis, samples were arranged in 96-well PCR plates so that comparisons could be made between PBMCs from infected cows and PBMCs from control cows, each stimulated with M. avium subsp. paratuberculosis or PBS (nil stimulation), on the same plate. The data are means ± standard errors of the means for 2 −ΔΔCt values for three or four infected cows and three or four control cows for each gene.

    Journal: Infection and Immunity

    Article Title: Evidence for a Novel Gene Expression Program in Peripheral Blood Mononuclear Cells from Mycobacterium avium subsp. paratuberculosis-Infected Cattle

    doi: 10.1128/IAI.71.11.6487-6498.2003

    Figure Lengend Snippet: Q-RT-PCR validation of gene expression changes observed following microarray analysis of PBMCs from control and infected cows. Genes to be validated were selected from a list of the genes whose expression was most significantly different following nil stimulation of PBMCs from infected and control cows. Q-RT-PCR was performed as described in Materials and Methods, and data were analyzed by using the 2 −ΔΔCt method with β-actin as the control gene. Mean values for control cow PBMCs with nil stimulation or M. avium subsp. paratuberculosis (MPTb) stimulation were used as calibrators for calculation of all 2 −ΔΔCt values, so that the values for control cows always bracketed 1.0. For Q-RT-PCR analysis, samples were arranged in 96-well PCR plates so that comparisons could be made between PBMCs from infected cows and PBMCs from control cows, each stimulated with M. avium subsp. paratuberculosis or PBS (nil stimulation), on the same plate. The data are means ± standard errors of the means for 2 −ΔΔCt values for three or four infected cows and three or four control cows for each gene.

    Article Snippet: However, stimulation of PBMCs with M. avium subsp. paratuberculosis caused a marked reduction in ALG-4 gene expression in infected cow PBMCs, resulting in a mean level of expression that was twofold lower than that in similarly treated control cow PBMCs (Fig. ).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Microarray, Infection, Polymerase Chain Reaction

    Q-RT-PCR validation of cDNA microarray results for PBMCs from control cows. Genes encoding Sentrin-(SUMO-1), MMP 1, and MMP 23 were selected for Q-RT-PCR validation from among the genes that exhibit differential expression in nil-stimulated and M. avium subsp. paratuberculosis -stimulated PBMCs from control uninfected cows. The gene encoding Sentrin-(SUMO-1) was selected as a representative of genes that exhibit up-regulation in control cow PBMCs stimulated with M. avium subsp. paratuberculosis compared to expression in nil-stimulated cells. The genes encoding MMP 1 and MMP 23 were selected because each was apparently down-regulated by M. avium subsp. paratuberculosis stimulation of control cow PBMCs on cDNA microarrays and because of previous data suggesting that MMP gene regulation is a major and consistent effect of M. avium subsp. paratuberculosis ). For these reasons, an analysis of nil-stimulated and M. avium subsp. paratuberculosis -stimulated PBMCs from infected cows was also included in this study. Q-RT-PCR was conducted as described in Materials and Methods by using gene-specific primers. Data were analyzed by using the 2 −ΔΔCt ) with β-actin as the control gene and nil stimulation within animal as the calibrator. The data are the means ± standard errors of the means for independent results from four infected cows and three control cows. MPTb, M. avium subsp. paratuberculosis .

    Journal: Infection and Immunity

    Article Title: Evidence for a Novel Gene Expression Program in Peripheral Blood Mononuclear Cells from Mycobacterium avium subsp. paratuberculosis-Infected Cattle

    doi: 10.1128/IAI.71.11.6487-6498.2003

    Figure Lengend Snippet: Q-RT-PCR validation of cDNA microarray results for PBMCs from control cows. Genes encoding Sentrin-(SUMO-1), MMP 1, and MMP 23 were selected for Q-RT-PCR validation from among the genes that exhibit differential expression in nil-stimulated and M. avium subsp. paratuberculosis -stimulated PBMCs from control uninfected cows. The gene encoding Sentrin-(SUMO-1) was selected as a representative of genes that exhibit up-regulation in control cow PBMCs stimulated with M. avium subsp. paratuberculosis compared to expression in nil-stimulated cells. The genes encoding MMP 1 and MMP 23 were selected because each was apparently down-regulated by M. avium subsp. paratuberculosis stimulation of control cow PBMCs on cDNA microarrays and because of previous data suggesting that MMP gene regulation is a major and consistent effect of M. avium subsp. paratuberculosis ). For these reasons, an analysis of nil-stimulated and M. avium subsp. paratuberculosis -stimulated PBMCs from infected cows was also included in this study. Q-RT-PCR was conducted as described in Materials and Methods by using gene-specific primers. Data were analyzed by using the 2 −ΔΔCt ) with β-actin as the control gene and nil stimulation within animal as the calibrator. The data are the means ± standard errors of the means for independent results from four infected cows and three control cows. MPTb, M. avium subsp. paratuberculosis .

    Article Snippet: However, stimulation of PBMCs with M. avium subsp. paratuberculosis caused a marked reduction in ALG-4 gene expression in infected cow PBMCs, resulting in a mean level of expression that was twofold lower than that in similarly treated control cow PBMCs (Fig. ).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Microarray, Expressing, Infection

    Identification of numerous gene expression changes in both nil-stimulated and M. avium subsp. paratuberculosis -stimulated PBMCs when expression levels were compared across infection groups by using a mixed-model analysis. Data from microarray analysis of PBMCs from six infected cows and four control cows were combined and analyzed as described in Materials and Methods by using a two-stage mixed model in SAS. The resulting least square (LS) means were used to construct interaction tables containing relative expression information and confidence intervals for each gene on the BOTL-3 cDNA microarray. Data were imported into Excel, and the Data Filter command was used to select genes with various expression differences (fold changes) and significance values ( P

    Journal: Infection and Immunity

    Article Title: Evidence for a Novel Gene Expression Program in Peripheral Blood Mononuclear Cells from Mycobacterium avium subsp. paratuberculosis-Infected Cattle

    doi: 10.1128/IAI.71.11.6487-6498.2003

    Figure Lengend Snippet: Identification of numerous gene expression changes in both nil-stimulated and M. avium subsp. paratuberculosis -stimulated PBMCs when expression levels were compared across infection groups by using a mixed-model analysis. Data from microarray analysis of PBMCs from six infected cows and four control cows were combined and analyzed as described in Materials and Methods by using a two-stage mixed model in SAS. The resulting least square (LS) means were used to construct interaction tables containing relative expression information and confidence intervals for each gene on the BOTL-3 cDNA microarray. Data were imported into Excel, and the Data Filter command was used to select genes with various expression differences (fold changes) and significance values ( P

    Article Snippet: However, stimulation of PBMCs with M. avium subsp. paratuberculosis caused a marked reduction in ALG-4 gene expression in infected cow PBMCs, resulting in a mean level of expression that was twofold lower than that in similarly treated control cow PBMCs (Fig. ).

    Techniques: Expressing, Infection, Microarray, Construct

    ATRA does not affect apoptosis of HDM-expanded Th2 cells. CTV labeled PBMCs from allergic asthmatic subjects were cultured with HDM Ag for 7d with ATRA or DMSO vehicle control. Annexin V staining was measured in the CTV low (unprolferated) and CTV high (proliferated HDM Ag specific) cells, after first gating on CD4+, 7AAD- lymphocytes. Above, data from a representative experiment. Below, combined results from 3 independent experiments. The percentage and number of annexin V positive cells are plotted. No statistical differences were observed by Student’s t test.

    Journal: Clinical and Molecular Allergy : CMA

    Article Title: The retinoic acid receptor-? modulators ATRA and Ro415253 reciprocally regulate human IL-5+ Th2 cell proliferation and cytokine expression

    doi: 10.1186/1476-7961-11-4

    Figure Lengend Snippet: ATRA does not affect apoptosis of HDM-expanded Th2 cells. CTV labeled PBMCs from allergic asthmatic subjects were cultured with HDM Ag for 7d with ATRA or DMSO vehicle control. Annexin V staining was measured in the CTV low (unprolferated) and CTV high (proliferated HDM Ag specific) cells, after first gating on CD4+, 7AAD- lymphocytes. Above, data from a representative experiment. Below, combined results from 3 independent experiments. The percentage and number of annexin V positive cells are plotted. No statistical differences were observed by Student’s t test.

    Article Snippet: Abbreviations RA: Retinoic acid; ATRA: All-trans retinoic acid; RARα: Retinoic acid receptor alpha; RXR: Retinoid X receptor; PBMC: Peripheral blood mononuclear cells; HDM: House dust mite; Ag: Antigen; APC: Antigen presenting cells; TCR: T cell receptor; CTV: Cell trace violet; Th2: T helper 2; Ro41: Ro41-5253; RARE: Retinoic acid response element; IL5p: Interleukin 5 gene promoter; IL13p: Interleukin 13 gene promoter; IL4p: Interleukin 4 gene promoter; mRNA: Messenger ribonucleic acid; ICCS: Intracellular cytokine staining; qRT-PCR: Quantitative real time PCR; DMSO: Dimethyl sulfoxide; DMEM: Dulbecco modified eagles medium; FBS: Fetal bovine serum; ANOVA: Analysis of variation; MFI: Mean fluorescence intensity; SEM: Standard error of mean

    Techniques: Labeling, Cell Culture, Staining

    Enhancement of Th2 cell output in HDM-stimulated PBMC cultures is reciprocally regulated by RARα modulators. Cell trace violet (CTV)-labeled PBMCs from allergic asthmatic subjects were stimulated with HDM Ag extract (40 U/ml) for 10d in the presence of ATRA (RARα agonist), Ro41 (RARα antagonist), or DMSO vehicle control. After 10d cultures were restimulated with PMA and ionomycin and ICCS was performed. (A) Representative flow plots are shown. Combined results from 3 cultures showing (B) the frequency of CTV low cells, after gating on viable HDM proliferated CD3+, CD4+, CD8- cells. Combined results from 3 cultures showing (C) the cell number and (D) percentage of Th2 cytokine producing HDM-expanded T cells. (E) Representative flow plots showing IL-13 vs. IL-5 expression after gating on viable HDM proliferated CD3 + CD4 + CTV low cells. Combined results from 3 cultures showing (F) cell number and (G) percentage of of IL-5 + (IL-5 + IL-13 + ) and IL-5 - (IL-5 - IL-13 + ) Th2 cell populations. Data points represent independent HDM-stimulated PBMC cultures. P-values were generated with 2-way ANOVA. In B and C, only 2 experiments were performed using IL-4 as an analyte.

    Journal: Clinical and Molecular Allergy : CMA

    Article Title: The retinoic acid receptor-? modulators ATRA and Ro415253 reciprocally regulate human IL-5+ Th2 cell proliferation and cytokine expression

    doi: 10.1186/1476-7961-11-4

    Figure Lengend Snippet: Enhancement of Th2 cell output in HDM-stimulated PBMC cultures is reciprocally regulated by RARα modulators. Cell trace violet (CTV)-labeled PBMCs from allergic asthmatic subjects were stimulated with HDM Ag extract (40 U/ml) for 10d in the presence of ATRA (RARα agonist), Ro41 (RARα antagonist), or DMSO vehicle control. After 10d cultures were restimulated with PMA and ionomycin and ICCS was performed. (A) Representative flow plots are shown. Combined results from 3 cultures showing (B) the frequency of CTV low cells, after gating on viable HDM proliferated CD3+, CD4+, CD8- cells. Combined results from 3 cultures showing (C) the cell number and (D) percentage of Th2 cytokine producing HDM-expanded T cells. (E) Representative flow plots showing IL-13 vs. IL-5 expression after gating on viable HDM proliferated CD3 + CD4 + CTV low cells. Combined results from 3 cultures showing (F) cell number and (G) percentage of of IL-5 + (IL-5 + IL-13 + ) and IL-5 - (IL-5 - IL-13 + ) Th2 cell populations. Data points represent independent HDM-stimulated PBMC cultures. P-values were generated with 2-way ANOVA. In B and C, only 2 experiments were performed using IL-4 as an analyte.

    Article Snippet: Abbreviations RA: Retinoic acid; ATRA: All-trans retinoic acid; RARα: Retinoic acid receptor alpha; RXR: Retinoid X receptor; PBMC: Peripheral blood mononuclear cells; HDM: House dust mite; Ag: Antigen; APC: Antigen presenting cells; TCR: T cell receptor; CTV: Cell trace violet; Th2: T helper 2; Ro41: Ro41-5253; RARE: Retinoic acid response element; IL5p: Interleukin 5 gene promoter; IL13p: Interleukin 13 gene promoter; IL4p: Interleukin 4 gene promoter; mRNA: Messenger ribonucleic acid; ICCS: Intracellular cytokine staining; qRT-PCR: Quantitative real time PCR; DMSO: Dimethyl sulfoxide; DMEM: Dulbecco modified eagles medium; FBS: Fetal bovine serum; ANOVA: Analysis of variation; MFI: Mean fluorescence intensity; SEM: Standard error of mean

    Techniques: Labeling, Flow Cytometry, Expressing, Generated

    C(−280)NFAT-binding activity does not depend on AP-1 cooperativity and has a higher affinity than NFAT sites with AP-1 cooperativity. a, NFAT binding to C(−280)NFAT is independent of AP-1. AP-1 sequence failed to compete for binding by C(−280)NFAT in EMSA using human PBMC extracts and vice versa. C(−280)NFAT failed to compete against AP-1 binding. *C-NFAT probe, C(−280)NFAT; *AP-1 probe, AP-1 oligonucleotide. b, NFAT-binding complex does not contain AP-1 components. Abs to Jun-B or c-Fos when incubated with PBMC extracts in EMSAs do not alter binding to the C(−280)NFAT probe. c, NFAT DNA-binding kinetic analysis. NFAT binding to C(−280)NFAT is more stable than to NFAT:AP-1 sites from the human and murine IL-2 promoter in PBMC extracts. Excess competitor for the indicated sites was added and the duration of binding was measured by EMSA. Right panel, The absorbance (OD) of the binding complex after addition of excess competitor. d, NFAT promoter sequence comparison. C(−280)NFAT sequence has four adenosine bases (shadowed) in common when aligned with NFAT IL-2 promoter sequences. Underlined adenosine bases are critical for DNA binding as determined by EMSA ( e ). Sp = species; h = human; m = mouse; D = A,T; N = A,C,T,G; X = A,T,C; a = noncoding sequence; b = EMSA competitor. e, Mutation analysis of C(−280)NFAT to identify nucleotides important for DNA binding. Single-base pair substitutions (C↔A and G↔T) across the C(−280)NFAT sequence were introduced into the indicated nucleotide positions and the corresponding double-stranded oligonucleotides containing mutations were used as competitor sequence in EMSA.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Induction of the CTLA-4 Gene in Human Lymphocytes Is Dependent on NFAT Binding the Proximal Promoter

    doi:

    Figure Lengend Snippet: C(−280)NFAT-binding activity does not depend on AP-1 cooperativity and has a higher affinity than NFAT sites with AP-1 cooperativity. a, NFAT binding to C(−280)NFAT is independent of AP-1. AP-1 sequence failed to compete for binding by C(−280)NFAT in EMSA using human PBMC extracts and vice versa. C(−280)NFAT failed to compete against AP-1 binding. *C-NFAT probe, C(−280)NFAT; *AP-1 probe, AP-1 oligonucleotide. b, NFAT-binding complex does not contain AP-1 components. Abs to Jun-B or c-Fos when incubated with PBMC extracts in EMSAs do not alter binding to the C(−280)NFAT probe. c, NFAT DNA-binding kinetic analysis. NFAT binding to C(−280)NFAT is more stable than to NFAT:AP-1 sites from the human and murine IL-2 promoter in PBMC extracts. Excess competitor for the indicated sites was added and the duration of binding was measured by EMSA. Right panel, The absorbance (OD) of the binding complex after addition of excess competitor. d, NFAT promoter sequence comparison. C(−280)NFAT sequence has four adenosine bases (shadowed) in common when aligned with NFAT IL-2 promoter sequences. Underlined adenosine bases are critical for DNA binding as determined by EMSA ( e ). Sp = species; h = human; m = mouse; D = A,T; N = A,C,T,G; X = A,T,C; a = noncoding sequence; b = EMSA competitor. e, Mutation analysis of C(−280)NFAT to identify nucleotides important for DNA binding. Single-base pair substitutions (C↔A and G↔T) across the C(−280)NFAT sequence were introduced into the indicated nucleotide positions and the corresponding double-stranded oligonucleotides containing mutations were used as competitor sequence in EMSA.

    Article Snippet: To study the dependence of CTLA-4 expression on NFAT activity in PBMCs, cell-permeable inhibitors that affect NFAT specifically were added to PBMCs and CTLA-4 gene expression was measured.

    Techniques: Binding Assay, Activity Assay, Sequencing, Incubation, Mutagenesis

    Identifying NFAT as the factor that binds to the proximal CTLA-4 promoter. a, Sequence of the proximal CTLA-4 promoter important for transcription and potential transcription factor recognition motifs predicted by Transcription ESS is shown from nucleotides −264 to −297 from the translational start site. This sequence has consensus sites for NFAT, c-Myb, and SRE and is defined as C(−280)NFAT. b, NFAT sequences compete away DNA binding to C(−280)NFAT probe. EMSA results with γ - 32 P-labeled dsC(−280)NFAT probe, with extracts prepared from stimulated human PBMCs. Competition assays were performed using excess double-stranded unlabeled oligonucleotides for C(−280)NFAT = C-NFAT, human IL-2 NFAT = hNFAT, Sp-1, or GAS sites. c, C(−280)NFAT oligonucleotide interacts with DNA-binding activity for NFAT and is competed by other NFAT sequences (hIL-2 NFAT and mIL-2 NFAT). Competition with excess c-Myb or SRE oligonucleotide does not alter DNA binding to the C(−280)NFAT probe. d, NFAT1 binds to C(−280)NFAT. EMSA performed in the presence of Abs specific to NFAT1 yields a supershifted band (arrow) whereas nonspecific Ab (Ns Ab) or anti-NFAT2 does not.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Induction of the CTLA-4 Gene in Human Lymphocytes Is Dependent on NFAT Binding the Proximal Promoter

    doi:

    Figure Lengend Snippet: Identifying NFAT as the factor that binds to the proximal CTLA-4 promoter. a, Sequence of the proximal CTLA-4 promoter important for transcription and potential transcription factor recognition motifs predicted by Transcription ESS is shown from nucleotides −264 to −297 from the translational start site. This sequence has consensus sites for NFAT, c-Myb, and SRE and is defined as C(−280)NFAT. b, NFAT sequences compete away DNA binding to C(−280)NFAT probe. EMSA results with γ - 32 P-labeled dsC(−280)NFAT probe, with extracts prepared from stimulated human PBMCs. Competition assays were performed using excess double-stranded unlabeled oligonucleotides for C(−280)NFAT = C-NFAT, human IL-2 NFAT = hNFAT, Sp-1, or GAS sites. c, C(−280)NFAT oligonucleotide interacts with DNA-binding activity for NFAT and is competed by other NFAT sequences (hIL-2 NFAT and mIL-2 NFAT). Competition with excess c-Myb or SRE oligonucleotide does not alter DNA binding to the C(−280)NFAT probe. d, NFAT1 binds to C(−280)NFAT. EMSA performed in the presence of Abs specific to NFAT1 yields a supershifted band (arrow) whereas nonspecific Ab (Ns Ab) or anti-NFAT2 does not.

    Article Snippet: To study the dependence of CTLA-4 expression on NFAT activity in PBMCs, cell-permeable inhibitors that affect NFAT specifically were added to PBMCs and CTLA-4 gene expression was measured.

    Techniques: Sequencing, Binding Assay, Labeling, Activity Assay

    Total percentages of (A) IFN-γ, (B) IL-10 and (C) IL-4 producing lymphocytes in neonates ( n = 18), foals until 12 weeks of age ( n = 15) and in adult horses ( n = 15). PBMC were stimulated with PMA and ionomycin. The cells were stained for intracellular cytokine production and were analyzed by flow cytometry. The percentages include all cells producing the respective cytokine including double positive cells. The horizontal lines within the data sets represent medians. *** p

    Journal: Veterinary Research

    Article Title: Interferon-gamma, interleukin-4 and interleukin-10 production by T helper cells reveals intact Th1 and regulatory TR1 cell activation and a delay of the Th2 cell response in equine neonates and foals

    doi: 10.1051/vetres/2010019

    Figure Lengend Snippet: Total percentages of (A) IFN-γ, (B) IL-10 and (C) IL-4 producing lymphocytes in neonates ( n = 18), foals until 12 weeks of age ( n = 15) and in adult horses ( n = 15). PBMC were stimulated with PMA and ionomycin. The cells were stained for intracellular cytokine production and were analyzed by flow cytometry. The percentages include all cells producing the respective cytokine including double positive cells. The horizontal lines within the data sets represent medians. *** p

    Article Snippet: Foal PBMC also showed a clear up-regulation of IFN-γ mRNA, a decreased IL-4 gene expression and greater IFN-γ/IL-4 transcript ratios in response to R. equi infection compared to adult horses [ ].

    Techniques: Staining, Flow Cytometry, Cytometry

    Flow cytometric analysis of IFN-γ, IL-4 and IL-10 producing PBMC after stimulation with PMA and ionomycin. PBMC were incubated in the presence of the secretion blocker Brefeldin A. Then, they were fixed and intracellular cytokine staining was performed. (A) Non-stimulated PBMC from a 6 week old foal after 4 h of incubation in medium with Brefeldin A. The left plot shows the gating (R1) on peripheral blood lymphocytes that was used for analysis of the data. The remaining three plots show a two-color staining of non-stimulated lymphocytes using anti-CD4 and different anti-cytokine antibodies. Typically, cytokines were not detected in equine peripheral blood lymphocytes in the absence of stimulation. Isotype controls for cytokine staining generally resulted in less than 0.05% of detectable cells. (B and C): PBMC from 4 adult horses were stimulated with PMA and ionomycin for up to 24 h and cytokine expression was measured at various time points. (B) Total percentages of IFN-γ, IL-10 and IL-4 producing cells in the lymphocyte population, (C) Percentages of IFN-γ + /IL-10 + , IFN-γ + /IL-10 − and IFN-γ − /IL-10 + cells during 24 h of stimulation. The data in B and C represent means and standard deviations. Differences in cytokine expression from one to the next time point were compared by Student’s t -tests. ** p = 0.001 to

    Journal: Veterinary Research

    Article Title: Interferon-gamma, interleukin-4 and interleukin-10 production by T helper cells reveals intact Th1 and regulatory TR1 cell activation and a delay of the Th2 cell response in equine neonates and foals

    doi: 10.1051/vetres/2010019

    Figure Lengend Snippet: Flow cytometric analysis of IFN-γ, IL-4 and IL-10 producing PBMC after stimulation with PMA and ionomycin. PBMC were incubated in the presence of the secretion blocker Brefeldin A. Then, they were fixed and intracellular cytokine staining was performed. (A) Non-stimulated PBMC from a 6 week old foal after 4 h of incubation in medium with Brefeldin A. The left plot shows the gating (R1) on peripheral blood lymphocytes that was used for analysis of the data. The remaining three plots show a two-color staining of non-stimulated lymphocytes using anti-CD4 and different anti-cytokine antibodies. Typically, cytokines were not detected in equine peripheral blood lymphocytes in the absence of stimulation. Isotype controls for cytokine staining generally resulted in less than 0.05% of detectable cells. (B and C): PBMC from 4 adult horses were stimulated with PMA and ionomycin for up to 24 h and cytokine expression was measured at various time points. (B) Total percentages of IFN-γ, IL-10 and IL-4 producing cells in the lymphocyte population, (C) Percentages of IFN-γ + /IL-10 + , IFN-γ + /IL-10 − and IFN-γ − /IL-10 + cells during 24 h of stimulation. The data in B and C represent means and standard deviations. Differences in cytokine expression from one to the next time point were compared by Student’s t -tests. ** p = 0.001 to

    Article Snippet: Foal PBMC also showed a clear up-regulation of IFN-γ mRNA, a decreased IL-4 gene expression and greater IFN-γ/IL-4 transcript ratios in response to R. equi infection compared to adult horses [ ].

    Techniques: Flow Cytometry, Incubation, Staining, Expressing

    Ratios of (A) IFN-γ + /IL-4 + and (B) IFN-γ + /IL-10 + lymphocytes in foals up to 3 months of age and adult horses. PBMC were stimulated with PMA and ionomycin. The cells were stained for intracellular cytokine production and measured by flow cytometry. The total percentages of IFN-γ, IL-4 and IL-10 producing lymphocytes were determined and ratios were calculated for the respective cytokines. The horizontal lines within the data points show the medians. *** p

    Journal: Veterinary Research

    Article Title: Interferon-gamma, interleukin-4 and interleukin-10 production by T helper cells reveals intact Th1 and regulatory TR1 cell activation and a delay of the Th2 cell response in equine neonates and foals

    doi: 10.1051/vetres/2010019

    Figure Lengend Snippet: Ratios of (A) IFN-γ + /IL-4 + and (B) IFN-γ + /IL-10 + lymphocytes in foals up to 3 months of age and adult horses. PBMC were stimulated with PMA and ionomycin. The cells were stained for intracellular cytokine production and measured by flow cytometry. The total percentages of IFN-γ, IL-4 and IL-10 producing lymphocytes were determined and ratios were calculated for the respective cytokines. The horizontal lines within the data points show the medians. *** p

    Article Snippet: Foal PBMC also showed a clear up-regulation of IFN-γ mRNA, a decreased IL-4 gene expression and greater IFN-γ/IL-4 transcript ratios in response to R. equi infection compared to adult horses [ ].

    Techniques: Staining, Flow Cytometry, Cytometry

    Flow cytometric analysis of CD4 + and CD8 + IFN-γ producing lymphocytes. PBMC from foals of different age groups and from adult horses were stimulated with PMA and ionomycin. The cells were stained for intracellular IFN-γ and cell surface CD4 or CD8 expression. (A) Flow cytometric analysis of one representative horse per age group. (B) Relative percentages of CD4 + /IFN-γ + and (C) CD8 + /IFN-γ + T cells in foals and adult horses within the IFN-γ producing cells (= 100%). (D) Percentages of IFN-γ + /IL-10 + lymphocytes in foals and adult horses. (E) A tri-color staining was performed on stimulated PBMC to confirm that the majority of the IFN-γ + /IL-10 + cells are CD4 + T cells. Gates were set on lymphocytes (see Fig. 1A) and on IFN-γ + cells (left panel). The IFN-γ + lymphocytes were then analyzed for IL-10 and CD4 staining (right panel). The horizontal lines within the data sets B, C and D represent medians. *** p

    Journal: Veterinary Research

    Article Title: Interferon-gamma, interleukin-4 and interleukin-10 production by T helper cells reveals intact Th1 and regulatory TR1 cell activation and a delay of the Th2 cell response in equine neonates and foals

    doi: 10.1051/vetres/2010019

    Figure Lengend Snippet: Flow cytometric analysis of CD4 + and CD8 + IFN-γ producing lymphocytes. PBMC from foals of different age groups and from adult horses were stimulated with PMA and ionomycin. The cells were stained for intracellular IFN-γ and cell surface CD4 or CD8 expression. (A) Flow cytometric analysis of one representative horse per age group. (B) Relative percentages of CD4 + /IFN-γ + and (C) CD8 + /IFN-γ + T cells in foals and adult horses within the IFN-γ producing cells (= 100%). (D) Percentages of IFN-γ + /IL-10 + lymphocytes in foals and adult horses. (E) A tri-color staining was performed on stimulated PBMC to confirm that the majority of the IFN-γ + /IL-10 + cells are CD4 + T cells. Gates were set on lymphocytes (see Fig. 1A) and on IFN-γ + cells (left panel). The IFN-γ + lymphocytes were then analyzed for IL-10 and CD4 staining (right panel). The horizontal lines within the data sets B, C and D represent medians. *** p

    Article Snippet: Foal PBMC also showed a clear up-regulation of IFN-γ mRNA, a decreased IL-4 gene expression and greater IFN-γ/IL-4 transcript ratios in response to R. equi infection compared to adult horses [ ].

    Techniques: Flow Cytometry, Staining, Expressing