Journal: International journal of molecular sciences

Article Title: NAC1/ACOX2 Axis as a Novel Therapeutic Target for Endometriosis-Related Ovarian Neoplasms.

doi: 10.3390/ijms26104938

Figure Lengend Snippet: Figure 1. Immunohistochemistry of NAC1 and ACOX2 in ERON surgical specimens. Representa- tive cases showing negative (0), weak (1+), moderate (2+), and strong (3+) staining intensities are presented for NAC1 (A) and ACOX2 (B). Images were taken from three different plots for each expression category. The correlation between NAC1 and ACOX2 expression was evaluated via Fisher’s exact test (C).

Article Snippet: The slides were then incubated overnight at 4 ◦C with antibodies against a monoclonal anti-NAC1 (9.27) antibody [52] or polyclonal anti-ACOX2 (Atlas Antibodies, Stockholm, Sweden) at a dilution of 1:200.

Techniques: Immunohistochemistry, Staining, Expressing

Journal: International journal of molecular sciences

Article Title: NAC1/ACOX2 Axis as a Novel Therapeutic Target for Endometriosis-Related Ovarian Neoplasms.

doi: 10.3390/ijms26104938

Figure Lengend Snippet: Figure 2. Prognostic significance of NAC1 and ACOX2 expressions in patients with ERONs. Kaplan– Meier survival analyses are shown for high NAC1/low ACOX2 expression in relation to progression- free survival (PFS) (A) and overall survival (OS) (B). M: months.

Article Snippet: The slides were then incubated overnight at 4 ◦C with antibodies against a monoclonal anti-NAC1 (9.27) antibody [52] or polyclonal anti-ACOX2 (Atlas Antibodies, Stockholm, Sweden) at a dilution of 1:200.

Techniques: Expressing

Journal: International journal of molecular sciences

Article Title: NAC1/ACOX2 Axis as a Novel Therapeutic Target for Endometriosis-Related Ovarian Neoplasms.

doi: 10.3390/ijms26104938

Figure Lengend Snippet: Figure 3. Pearson’s correlation between NAC1 and ACOX2 expression in OCCC cells. Representative results of Western blot analysis of NAC1 and ACOX2 in each OCCC cell line (A) and their correlation by quantitative evaluation (B) measured by ImageJ software (version 1.53k). The full-length Western blot gel image is available in Supplementary Figure S2.

Article Snippet: The slides were then incubated overnight at 4 ◦C with antibodies against a monoclonal anti-NAC1 (9.27) antibody [52] or polyclonal anti-ACOX2 (Atlas Antibodies, Stockholm, Sweden) at a dilution of 1:200.

Techniques: Expressing, Western Blot, Software

Journal: International journal of molecular sciences

Article Title: NAC1/ACOX2 Axis as a Novel Therapeutic Target for Endometriosis-Related Ovarian Neoplasms.

doi: 10.3390/ijms26104938

Figure Lengend Snippet: Figure 4. siRNA-knockdown analysis of NAC1 in OCCC cell lines. Western blotting was used to evaluate NAC1 and ACOX2 protein expressions in OV207 and KF28 OCCC cell lines after siRNA knockdown (A,B). NAC1 knockdown was confirmed in both cell lines (C,D), and efficient knockdown of NAC1 led to significant upregulation of ACOX2 in both cell lines (E,F). The full-length Western blot gel image is available in Supplementary Figure S3.

Article Snippet: The slides were then incubated overnight at 4 ◦C with antibodies against a monoclonal anti-NAC1 (9.27) antibody [52] or polyclonal anti-ACOX2 (Atlas Antibodies, Stockholm, Sweden) at a dilution of 1:200.

Techniques: Knockdown, Western Blot

Journal: International journal of molecular sciences

Article Title: NAC1/ACOX2 Axis as a Novel Therapeutic Target for Endometriosis-Related Ovarian Neoplasms.

doi: 10.3390/ijms26104938

Figure Lengend Snippet: Figure 5. Transcriptional activity of the ACOX2 promoter in OCCC cell lines with different levels of NAC1 expression. The promoter sequence of the ACOX2 gene spans from −1488 to +72 upstream of the transcriptional start site (arrowhead) (A). The underlines indicate the 6 CATG consensus motifs for NAC1 binding. (B) The luciferase (LUC) reporter construct, in which the ACOX2 full-length promoter was inserted into the pGL3 basic vector, was prepared (B) and applied to the luciferase reporter assays. The transcriptional activity of the ACOX2 promoter was evaluated in each reporter construct using RK3E cells lacking endogenous expression of NAC1 (C) or those with NAC1 overexpression (D). The promoter activity of ACOX2 was further evaluated in OV207 and KF28 cells, both expressing endogenous NAC1 expression (E,F). NAC1 in each cell type was then knocked down by siRNA, and the promoter activity of ACOX2 was also evaluated (G,H). Each experiment was independently repeated three times with three technical replicates. Statistical significance is represented as * p < 0.05; ** p < 0.005.

Article Snippet: The slides were then incubated overnight at 4 ◦C with antibodies against a monoclonal anti-NAC1 (9.27) antibody [52] or polyclonal anti-ACOX2 (Atlas Antibodies, Stockholm, Sweden) at a dilution of 1:200.

Techniques: Activity Assay, Expressing, Sequencing, Binding Assay, Luciferase, Construct, Plasmid Preparation, Over Expression

Journal: International journal of molecular sciences

Article Title: NAC1/ACOX2 Axis as a Novel Therapeutic Target for Endometriosis-Related Ovarian Neoplasms.

doi: 10.3390/ijms26104938

Figure Lengend Snippet: Figure 6. Promoter assays to identify cis-elements of the ACOX2 promoter responsible for NAC1 repression. (A,B) The full-length or 5′ deleted promoter sequences containing different numbers of CATG sites were prepared and incorporated into the luciferase reporter constructs, followed by luciferase assays using OV207 and KF28 cells. Furthermore, mutant reporter plasmids, in which the most proximal CATG sites were substitution-mutated into the full-length promoter or 5′ deleted promoter containing only this site, were prepared (C,D), followed by luciferase assays using both cell lines. WT: wild type CATG site. MT: substitution mutated CATG site. Each experiment was indepen- dently repeated three times with three technical replicates. Statistical significance is represented as * p < 0.05; ** p < 0.005. NS: Not significant.

Article Snippet: The slides were then incubated overnight at 4 ◦C with antibodies against a monoclonal anti-NAC1 (9.27) antibody [52] or polyclonal anti-ACOX2 (Atlas Antibodies, Stockholm, Sweden) at a dilution of 1:200.

Techniques: Luciferase, Construct, Mutagenesis

Journal: International journal of molecular sciences

Article Title: NAC1/ACOX2 Axis as a Novel Therapeutic Target for Endometriosis-Related Ovarian Neoplasms.

doi: 10.3390/ijms26104938

Figure Lengend Snippet: Figure 7. ChIP analysis to identify the binding of NAC1 to each CATG sequence on the ACOX2 promoter using OV207 and KF28 cells. Primer sets for quantitative PCRs targeting each CATG 1, 2, 3, 4, 5 site generated equivalent levels of immunoprecipitates by the NAC1 antibody compared to those with the control IgG in both cell lines (A–E). In contrast, primer sets targeting the most proximal CATG 6 site generated apparently increased levels of immunoprecipitates compared to those with control IgG in both cell lines (F). Normal rabbit IgG (IgG) served as a control antibody, while NAC1 antibody (Ab) was used to detect NAC1 binding. The data are shown as mean values relative to the input, expressed as a percentage (% of input). The error bars indicate the standard deviation, based on three independent measurements (n = 3). Statistical significance is represented as * p < 0.05; ** p < 0.005.

Article Snippet: The slides were then incubated overnight at 4 ◦C with antibodies against a monoclonal anti-NAC1 (9.27) antibody [52] or polyclonal anti-ACOX2 (Atlas Antibodies, Stockholm, Sweden) at a dilution of 1:200.

Techniques: Binding Assay, Sequencing, Generated, Control, Standard Deviation

Journal: International journal of molecular sciences

Article Title: NAC1/ACOX2 Axis as a Novel Therapeutic Target for Endometriosis-Related Ovarian Neoplasms.

doi: 10.3390/ijms26104938

Figure Lengend Snippet: Figure 8. Association between NAC1 expression and key fatty acid metabolism-related genes. Gene expression analysis demonstrates a significant reduction in stearoyl-coenzyme A desaturase 1 (SCD1) and fatty acid-binding protein 4 (FABP4) expression in NAC1 siRNA-treated cells compared to control siRNA-treated cells in OV207 and KF28 cell lines (A,B). The error bars indicate the standard deviation, based on three independent measurements (n = 3). Statistical significance is represented as * p < 0.05.

Article Snippet: The slides were then incubated overnight at 4 ◦C with antibodies against a monoclonal anti-NAC1 (9.27) antibody [52] or polyclonal anti-ACOX2 (Atlas Antibodies, Stockholm, Sweden) at a dilution of 1:200.

Techniques: Expressing, Gene Expression, Binding Assay, Control, Standard Deviation

Journal: International journal of molecular sciences

Article Title: NAC1/ACOX2 Axis as a Novel Therapeutic Target for Endometriosis-Related Ovarian Neoplasms.

doi: 10.3390/ijms26104938

Figure Lengend Snippet: Figure 9. Proposed model for the role of NAC1 in the development of ERONs via the regulation of ACOX2 expression.

Article Snippet: The slides were then incubated overnight at 4 ◦C with antibodies against a monoclonal anti-NAC1 (9.27) antibody [52] or polyclonal anti-ACOX2 (Atlas Antibodies, Stockholm, Sweden) at a dilution of 1:200.

Techniques: Expressing

Journal: Science Advances

Article Title: NAC1 modulates autoimmunity by suppressing regulatory T cell–mediated tolerance

doi: 10.1126/sciadv.abo0183

Figure Lengend Snippet: T cells from the thymus, peripheral LNs, and spleen of WT or NAC1 −/− mice were analyzed by flow cytometry and calculated for numbers or percentages. ( A ) CD4 and CD8 in the thymus. The DN populations were analyzed for DN1 to DN4 stages on the basis of CD44 and CD25. Data shown are the representative of five mice per group of three independent experiments. ( B ) Numbers and percentages of total thymocytes, CD4 or CD8 SP, and the percentages of DN2 or DN4 cells. Data shown are the representative of three identical experiments. The values represent means ± SD ( N = 4 or 5). ** P < 0.01, **** P < 0.0001, ns, no difference, Student’s unpaired t test. ( C ) CD4 and CD8 T cells from the pooled LNs and spleen. Data shown are the representative of five mice per group of three independent experiments. ( D ) Numbers and percentages of T cells from the pooled LNs and spleen. * P < 0.05, ** P < 0.01. ( E ) Representative CD4 + FoxP3 + T regs in the pooled LNs, spleen, blood, and thymus, gating on CD4 + populations. Data shown are the representative of three identical experiments ( N = 4 or 5). * P < 0.05, ** P < 0.01, **** P < 0.0001. ( F ) Numbers and percentages of T regs . Data shown are the representative of three identical experiments. The values represent the means ± SD ( N = 4 or 5). *** P < 0.001; ns, no statistical difference, Student’s unpaired t test.

Article Snippet: Rabbit NAC1 (no. 4183), HDAC6 (no. 7612), and β-actin (no. 8457) antibodies were purchased from Cell Signaling (Beverly, MA).

Techniques: Flow Cytometry

Journal: Science Advances

Article Title: NAC1 modulates autoimmunity by suppressing regulatory T cell–mediated tolerance

doi: 10.1126/sciadv.abo0183

Figure Lengend Snippet: The naive CD4 + CD25 − T cells from the pooled spleen and LNs of WT or NAC1 −/− mice were induced to iT regs in vitro in the presence of TGF-β for 5 days ( A to C ), or purified CD4 + T regs from the pooled LNs and spleen of WT or NAC1 −/− mice were stimulated with anti-CD3 plus CD28 antibodies in the presence of rIL-2 for various times ( D to F ). (A) Expression of NAC1 in naïve CD4 and iT regs of WT T cells detected by immunoblots. (B) Expression of CD25 and FoxP3 in iT regs generated from WT and NAC1 −/− CD4 + CD25 − T cells by flow cytometry. Data shown are the representative of three identical experiments. (C) MFI of FoxP3 in iT regs generated from WT and NAC1 −/− CD4 + CD25 − T cells as analyzed by flow cytometry. Results shown are the means ± SD of three identical experiments. ** P < 0.01, Student’s unpaired t test. (D) Cell proliferation/division by carboxyfluorescein diacetate succinimidyl ester (CFSE)–based flow cytometry. Data shown are the representative of three identical experiments. (E) Percentage and (F) numbers of cell recovery on various days were examined by trypan blue exclusion. The numbers of T cells present on day 0 were assigned a value of 100%, and numbers surviving on various days were used to calculate the percentage recovery relative to day 0. Data shown represent the means ± SEM of percentage change or numbers of three independent experiments. All P > 0.05, Student’s unpaired t test.

Article Snippet: Rabbit NAC1 (no. 4183), HDAC6 (no. 7612), and β-actin (no. 8457) antibodies were purchased from Cell Signaling (Beverly, MA).

Techniques: In Vitro, Purification, Expressing, Western Blot, Generated, Flow Cytometry, Cell Recovery

Journal: Science Advances

Article Title: NAC1 modulates autoimmunity by suppressing regulatory T cell–mediated tolerance

doi: 10.1126/sciadv.abo0183

Figure Lengend Snippet: Purified CD4 + T regs from the pooled LNs and spleen of WT or NAC1 −/− mice were stimulated with anti-CD3 plus CD28 antibodies in the presence of rIL-2 for various times. ( A ) OCR. ( B ) ECAR. ( C ) Cytokine production by intracellular staining. ( D ) Cytokine secretion by ELISA. ( E ) In vitro suppressive assay. ( F to I ) T cell transfer model of colitis. (F) Changes of body weight. (G) Representative hematoxylin and eosin (H&E)–stained sections of gut tissues. (H) T reg frequencies/numbers in the adoptive transfer–induced colitis model. (I and J to M ) Lipopolysaccharide (LPS)–mediated colitis. (I) Representative colon images of the LPS-induced colitis. (J) Colon lengths in LPS-induced colitis. (K) Representative H&E-stained sections of images of proximal colon cross section. (L and M) Numbers of transferred T regs and FoxP3 MFI (bottom), gating on Thy1.2 + populations (top). ( N to P ) In vivo analyses of T regs . WT and NAC1 −/− T regs (Thy1.2 + ) were labeled with CFSE and intravenously injected into mice. On days 2 and 5, the transferred Th1.2 + T regs were analyzed by flow cytometry. (N) Proliferation by CFSE. (M) FoxP3 expression. (P) Apoptosis. ( Q and R ) In vivo cotransfer of CFSE-labeled WT (Thy1.1 + ) and NAC1 −/− (Thy1.2 + ) at 1:1 ratio into recipients and examined the NAC1 −/− /WT ratios of T regs on days 2 and 5. Data shown are the means ± SD of three independent experiments and are the representative of three identical experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, Student’s unpaired t test.

Article Snippet: Rabbit NAC1 (no. 4183), HDAC6 (no. 7612), and β-actin (no. 8457) antibodies were purchased from Cell Signaling (Beverly, MA).

Techniques: Purification, Staining, Enzyme-linked Immunosorbent Assay, In Vitro, Adoptive Transfer Assay, In Vivo, Labeling, Injection, Flow Cytometry, Expressing

Journal: Science Advances

Article Title: NAC1 modulates autoimmunity by suppressing regulatory T cell–mediated tolerance

doi: 10.1126/sciadv.abo0183

Figure Lengend Snippet: WT or NAC1 −/− mice were challenged with either bovine type II collagen in complete Freund’s adjuvant by one intradermal immunization at two sites in the base and slightly above of the tail on day 0, or by oral ingestion of 3% DSS (MP Biomedicals) in drinking water for 5 days. ( A to C ) Arthritis: The histology of the joints (A), arthritis incidence (B), and clinical score (C) were evaluated by examining the paws. Values are the means ± SEM of three independent experiments ( n = 10). **** P < 0.0001 in (B) and (C), simple linear regression. ( D to H ) Colitis: The severity of colitis activity was graded on the designated dates. Histology of colon (D), animal body weight change (E), survival (F), animal size and colon length (G), and the resultant IBD disease activity index (H) were determined. Values are the means ± SEM of three independent experiments ( n = 10). **** P < 0.0001 in (E) and (H), simple linear regression. **** P < 0.0001 in (F), survival curve comparison ** P < 0.01.

Article Snippet: Rabbit NAC1 (no. 4183), HDAC6 (no. 7612), and β-actin (no. 8457) antibodies were purchased from Cell Signaling (Beverly, MA).

Techniques: Adjuvant, Activity Assay, Comparison

Journal: Science Advances

Article Title: NAC1 modulates autoimmunity by suppressing regulatory T cell–mediated tolerance

doi: 10.1126/sciadv.abo0183

Figure Lengend Snippet: Naive WT and NAC1 −/− CD4 + CD25 + T regs were purified and used in these analyses. ( A ) Diagram of mouse FoxP3 DNA methylation. Seven CpG sites of FoxP3 regulators (ADS657, ADS569, ADS1183, ADS442, ADS443, ADS779, and ADS1184) were analyzed. ( B ) Four CpG regions of FoxP3 regulators, including Distal Region (Regions ADS657 and ADS569), Proximal Region (ADS1183), CNS 2 Region (TSDR ADS443), and 3′ Downstream (ADS1184), were analyzed. ( C ) Genomic DNA of the lymphocytes from LNs and spleen of WT or NAC1 −/− mice was analyzed for methylation status of CNS2 (TSDR) and FoxP3 proximal promoter, respectively. The degree of methylation at each CpG motif is depicted according to the color code. A representative is shown. ( D ) Percentage of methylation of each FoxP3 CpG based on two experiments. P > 0.05; unpaired t test.

Article Snippet: Rabbit NAC1 (no. 4183), HDAC6 (no. 7612), and β-actin (no. 8457) antibodies were purchased from Cell Signaling (Beverly, MA).

Techniques: Purification, DNA Methylation Assay, Methylation

Journal: Science Advances

Article Title: NAC1 modulates autoimmunity by suppressing regulatory T cell–mediated tolerance

doi: 10.1126/sciadv.abo0183

Figure Lengend Snippet: ( Ai ) NAC1 ChIP-seq. Approximately 100 differential peaks (NAC1 versus input) were identified using HOMER. NAC1 peak density was not enriched within the regulatory elements of FoxP3. The genes targeted by NAC1-enriched islands in WT T regs were presented. Representative genomic regions (promotor and CNS1-3) show NAC1 enrichment. Normalized ChIP-seq reads (bigWig) and enriched islands (bed) are shown. ( Aii ) ATAC-seq. Differential accessibility at the FoxP3 gene locus in WT T regs compared with NAC1 −/− T regs is presented. There were more than 10,000 differential ATAC-seq peaks in total, but there were not any significant differential accessibility at the FoxP3 gene locus in WT T regs as compared with NAC1 −/− T regs . ( Aiii ) FoxP3 ChIP-seq. The genes targeted by FoxP3-enriched islands in WT or NAC1 −/− T regs are presented. ( B ) RNA sequencing. FoxP3 was not differentially expressed in NAC1 −/− T regs among ~200 differentially expressed genes, i.e., FoxP3 gene was identically expressed in WT T regs and NAC1 −/− T regs . ( C ) Volcano plot (left) and summarized mRNA change between WT and NAC1 −/− T regs (right). Black dots represent the significant changed genes (log 2 fold change >2, P < 0.05). Red dots represent the top 5 up-regulated genes in NAC1 −/− T regs , and blue dots represent the top 5 down-regulated genes in NAC1 −/− T regs compared to the WT. FoxP3 was marked as a green dot. All results shown are the representative of three identical experiments.

Article Snippet: Rabbit NAC1 (no. 4183), HDAC6 (no. 7612), and β-actin (no. 8457) antibodies were purchased from Cell Signaling (Beverly, MA).

Techniques: ChIP-sequencing, RNA Sequencing

Journal: Science Advances

Article Title: NAC1 modulates autoimmunity by suppressing regulatory T cell–mediated tolerance

doi: 10.1126/sciadv.abo0183

Figure Lengend Snippet: ( A ) NAC1 and FoxP3 in T regs from the FIR reporter mice by immunoblots. ( B ) NAC1 and FoxP3 mRNA of WT T regs generated in vitro and with ectopic expression of NAC1 by reverse transcription polymerase chain reaction. *** P < 0.001. ( C ) FoxP3 protein of the sorted T regs generated in vitro and with ectopic expression of NAC1 by immunoblots. ( D ) Immunofluorescence staining of 4′,6-diamidino-2-phenylindole, NAC1, and FoxP3 in T regs generated in vitro. ( E ) Expressions of FoxP3 and NAC1 in T regs from WT and NAC1 −/− mice by immunoblots. ( F ) FoxP3 was immunoprecipitated and examined for its acetylation of lysine. ( G ) WT T regs were cultured in the presence of various cytokines for 12 hours, and then the expressions of FoxP3 and NAC1 were analyzed by immunoblots. ( H ) FoxP3 from WT T regs treated in (G) was immunoprecipitated and examined for its acetylation. ( I ) WT or NAC1 −/− T regs were treated with cycloheximide (150 μg/ml) and chased for the indicated hours. FoxP3 protein level was analyzed by immunoblotting. ( J ) HDAC expression. ( K ) HDAC9 and FoxP3 of the sorted WT or NAC1 −/− T regs generated in vitro and with ectopic expression of either the control Mig vector or human HDAC9 vector were analyzed by immunoblots. All the data shown are the representative of three identical experiments. ( L ) Proposed model of regulation of FoxP3 by NAC1 in immunity.

Article Snippet: Rabbit NAC1 (no. 4183), HDAC6 (no. 7612), and β-actin (no. 8457) antibodies were purchased from Cell Signaling (Beverly, MA).

Techniques: Western Blot, Generated, In Vitro, Expressing, Reverse Transcription, Polymerase Chain Reaction, Immunofluorescence, Staining, Immunoprecipitation, Cell Culture, Control, Plasmid Preparation

Journal: bioRxiv

Article Title: NAC1 Modulates Autoimmunity by Suppressing Regulatory T Cell-Mediated Tolerance

doi: 10.1101/2022.03.01.482525

Figure Lengend Snippet: ( A ) Expressions of NAC1, FoxP3 and β -actin in Tregs from the FIR reporter mice were analyzed by immunoblots. CD4 + Tregs from the LNs and spleen of the Foxp3-IRES-mRFP (FIR) reporter mice, WT or NAC1 -/- mice, were analyzed. Data shown are the representative of three identical experiments. ( B ) NAC1 mRNA of WT Tregs generated in vitro and with ectopic expression of NAC1 were analyzed by RT-PCR. ***, P <0.001, Student’s unpaired t -test. Data shown are the representative of three identical experiments. ( C ) FoxP3 protein of the sorted Tregs generated in vitro and with ectopic expression of NAC1 were analyzed by immunoblots. Data shown are the representative of three identical experiments. ( D ) Immunofluorescent staining of DAPI, NAC1 and FoxP3 in Tregs generated in vitro . Data shown are the representative of three identical experiments. (E) Expressions of FoxP3, NAC1 and β -actin in Tregs from WT and NAC1 -/- mice were analyzed by immunoblots. Data shown are the representative of three identical experiments. ( F ) FoxP3 was immunoprecipitated from WT or NAC1 -/- Tregs and examined for its Acetylation of Lysine (Pan Acetylation). Acetylated FoxP3 (upper panel), IgG control (middle panel) and FoxP3 (lower panel) were examined by immunoblotting. Data shown are the representative of three identical experiments. ( G ) WT Tregs were cultured in the presence of TGF- β , IL-1 β or TFN- α for 12 hours, then the expressions of FoxP3, NAC1 and β -actin were analyzed by immunoblots. Data shown are the representative of three identical experiments. ( H ) WT Tregs were cultured in the presence of TGF- β 1 (2 ng/ml), IL- 1 β (10 ng/ml) or TFN- α (10 ng/ml) for 12 hours, then FoxP3 was immunoprecipitated and examined for its acetylation. Acetylated FoxP3 (upper panel), IgG control (middle panel), and FoxP3 (lower panel) were examined by immunoblots. Data shown are the representative of three identical experiments. ( I ) The pulse-chase experiments. WT or NAC1 -/- Tregs were treated with cycloheximide (150 μg/ml) for the indicated hours. FoxP3 protein level was analyzed by immunoblotting (upper panel); a plot indicated that with NAC1 deletion, the stability of FoxP3 was lifted in NAC1 -/- Tregs compared to the WT group (lower graph). Data shown are the representative of two identical experiments. (J) HDAC expression in WT and NAC1 -/- Tregs. Expression of HDAC6, HDAC9, HDAC11 and β -actin in WT and NAC1 -/- Tregs was analyzed by immunoblotting (upper panel). FoxP3 was immunoprecipitated and the immunoprecipitates examined for HDAC9 (lower panel). Data shown are the representative of two identical experiments. ( K ) Proposed model of regulation of FoxP3 by NAC1 in immunity.

Article Snippet: Rabbit NAC1 (#4183), HDAC6 (#7612) and β -actin (#8457) antibodies were purchased from Cell Signaling (Beverly, MA).

Techniques: Western Blot, Generated, In Vitro, Expressing, Reverse Transcription Polymerase Chain Reaction, Staining, Immunoprecipitation, Control, Cell Culture, Pulse Chase