dna constructs rela flag 22  (Addgene inc)

Journal: Science immunology

Article Title: A Stk4-Foxp3-NF-κB p65 transcriptional complex promotes T reg cell activation and homeostasis.

doi: 10.1126/sciimmunol.abl8357

Figure Lengend Snippet: Fig. 2. Stk3/4-deficient Treg showed transcriptome defects on TCR/p65/Foxp3-regulated genes. (A) Volcano plot of gene expression and (B) pathway analysis of gene transcripts using the KEGG database in YFP+CD4+ Treg cells from Foxp3YFPCre/+Stk3Δ/ΔStk4Δ/Δ versus Foxp3YFPCre mice. TH1, T helper 1. (C to E) Gene set enrichment of TCR/p65/Foxp3-dependent genes. (F) Heatmap of gene transcripts of YFP+CD4+ Treg cells isolated from Foxp3YFPCre (n = 7) and Foxp3YFPCre/+Stk3Δ/ΔStk4Δ/Δ (n = 6) mice. Counts were normalized using DESeq2-VST for heatmap visualization. (G) Heatmap showing the binding profile of the p65 ChIP-seq signal for Foxp3YFPCre, Foxp3YFPCreStk3Δ/ΔStk4Δ/Δ Treg, pooled input, and IgG isotype control samples at TSS and flanking ±2-kb region of the expressed genes. The Y axis indicates the number of counts per million mapped to denoted regions. (H) Venn diagram of the number of p65 pull-down ChIP-seq–enriched genes overlapping between WT Treg cells, Stk3/4-deficient Treg cells, and Foxp3-binding genes. (I) Arrows indicate the scatter plots of related gene transcript expression in RNA-seq (n = 7 for Foxp3YFPCre

Article Snippet: MycMST1 (#8847) (50), Flag-p65 (#20012) (51), pCL-Eco (#12371) (52), MSCV-IRES-Thy1.1 DEST (#17442), and MSCV-FOXP3IRES-Thy1.1 retroviral vector (#17443) (53) were from Addgene.

Techniques: Gene Expression, Isolation, Binding Assay, ChIP-sequencing, Control, Expressing, RNA Sequencing

Journal: Science immunology

Article Title: A Stk4-Foxp3-NF-κB p65 transcriptional complex promotes T reg cell activation and homeostasis.

doi: 10.1126/sciimmunol.abl8357

Figure Lengend Snippet: Fig. 3. TCR activation stimulates Stk4 nucleus translocation and colocalization with Foxp3. (A and B) Confocal microscopic analysis of CD4, Foxp3, STK4, DAPI, and merge (A) and ratios of nuclear/total Stk4 and frequencies of Stk4 and Foxp3 colocalization (B) in Foxp3YFPCre Treg cell cultures either unstimulated or stimulated with anti- CD3 + anti-CD28 mAbs as indicated (n = 10 per group). (C) Immunoblot (IB) analysis of Stk4 and Foxp3 association with p65 in Foxp3YFPCre Treg cells that were stimulated overnight with anti-CD3/CD28. Cell lysates were immunoprecipitated and immunoblotted with the indicated antibodies. (D and E) Immunoblot analysis of Stk4 and Foxp3 association with p65 in HEK293T cells transfected with the respective plasmids. Cell lysates were immunoprecipitated with anti-V5 mAb (specific for V5-tagged Foxp3) (D) or anti–c-Myc mAb (E) and then immunoblotted with the indicated antibodies. (F) densitometric analysis of immunoblots of p65 (top) and Foxp3 (bottom) shown in (E). (G and H) Confocal microscopic analysis (G) and frequencies (H) of CD4, Foxp3, STK4, DAPI, and merge and ratios of nuclear/total Stk4 and frequencies of Stk4 and Foxp3 colocalization in Foxp3YFPCre Treg cells either unstimulated or stimulated with anti-CD3 + anti-CD28 mAbs without or with the Stk4 kinase inhibitor XMU- MP-1 (n = 19 per group). (I) Stk4-p65 association is Stk4 kinase activity dependent. Immunoblot analysis of Stk4-p65 association in HEK293T cells transfected with plas- mids encoding either Stk4 kinase–competent (Stk4) or Stk4 kinase–deficient (Stk4K59R) proteins. Each point represents one cell for confocal studies and one blot for immunoblot analyses. Error bars indicate the SEM. Statistical tests: one-way ANOVA with posttest analysis (B and F), two-way ANOVA with posttest analysis (H), and Student’s unpaired two-tailed t test (C, D, and I). *P < 0.05, **P < 0.01, ***P < 0.005, and ****P < 0.0001.

Article Snippet: MycMST1 (#8847) (50), Flag-p65 (#20012) (51), pCL-Eco (#12371) (52), MSCV-IRES-Thy1.1 DEST (#17442), and MSCV-FOXP3IRES-Thy1.1 retroviral vector (#17443) (53) were from Addgene.

Techniques: Activation Assay, Translocation Assay, Western Blot, Immunoprecipitation, Transfection, Activity Assay, Two Tailed Test

Journal: Science immunology

Article Title: A Stk4-Foxp3-NF-κB p65 transcriptional complex promotes T reg cell activation and homeostasis.

doi: 10.1126/sciimmunol.abl8357

Figure Lengend Snippet: Fig. 4. Foxp3 S418 phosphorylation is Stk4 dependent and stabilizes the Stk4-Foxp3-p65 complex. (A and B) Immunoblot analysis (top) and densitometry (bottom) of p-serine/threonine Foxp3 (A) and Foxp3 p-S418 Foxp3 (B) in Foxp3YFPCre and Foxp3YFPCreStk3Δ/ΔStk4Δ/Δ Treg cells stimulated with anti-CD3/CD28 beads. Cell lysates were immunoprecipitated with anti-Foxp3 antibody and then immunoblotted with the indicated antibody. (C and D) Immunoblot analysis (top) and densi- tometry (bottom) of p-S418 Foxp3 in Jurkat cells that were transfected with the indicated plasmids and then stimulated with CD3/CD28 beads. Cell lysates were im- munoprecipitated with an anti-V5 mAb (C) or with anti-Flag mAb (D) and then immunoblotted with the indicated antibodies. (E and F) Immunoblot analysis (E) and densitometry (F) of p65 and Foxp3 association in HEK293T cells transfected with the indicated plasmids. Cell lysates were immunoprecipitated with anti-Flag mAb and then immunoblotted with the indicated antibodies. (G) Scatter plot representation of CD25 and Thy1.1 MFI in Foxp3YFPCre or in transfected Foxp3YFPCreStk3Δ/ΔStk4Δ/Δ Treg with the indicated retrovirus (n = 9 for each group). The results represent a pool of three independent experiments. Each point represents one blot for immunoblot analysis and one mouse. Error bars indicate SEM. Statistical tests: Student’s unpaired two-tailed t test (A, B, and D) and one-way ANOVAwith posttest analysis (C, F, and G). *P < 0.05, **P < 0.01, ***P < 0.005, and ****P < 0.0001.

Article Snippet: MycMST1 (#8847) (50), Flag-p65 (#20012) (51), pCL-Eco (#12371) (52), MSCV-IRES-Thy1.1 DEST (#17442), and MSCV-FOXP3IRES-Thy1.1 retroviral vector (#17443) (53) were from Addgene.

Techniques: Phospho-proteomics, Western Blot, Immunoprecipitation, Transfection, Two Tailed Test

Journal: Science immunology

Article Title: A Stk4-Foxp3-NF-κB p65 transcriptional complex promotes T reg cell activation and homeostasis.

doi: 10.1126/sciimmunol.abl8357

Figure Lengend Snippet: Fig. 5. Impaired p65 nuclear translocation and enhanced degradation in Stk3/4-deficient Treg cells. (A) Confocal microscopic analysis of CD4, Foxp3, p65, DAPI, and merge. (B) Ratios of nuclear/total Stk4 and p65/Foxp3 colocalization in Foxp3YFPCre Treg cells either unstimulated or stimulated with anti-CD3 + anti-CD28 mAbs at the indicated time points (n = 12 per group). (C and D) Immunoblot analysis (C) and densitometry (D) of p-p65, p65, and β-actin in Foxp3YFPCre and Foxp3YFPcreStk3Δ/ΔStk4Δ/Δ

Article Snippet: MycMST1 (#8847) (50), Flag-p65 (#20012) (51), pCL-Eco (#12371) (52), MSCV-IRES-Thy1.1 DEST (#17442), and MSCV-FOXP3IRES-Thy1.1 retroviral vector (#17443) (53) were from Addgene.

Techniques: Translocation Assay, Western Blot

Journal: Science immunology

Article Title: A Stk4-Foxp3-NF-κB p65 transcriptional complex promotes T reg cell activation and homeostasis.

doi: 10.1126/sciimmunol.abl8357

Figure Lengend Snippet: Fig. 6. Overexpression of p65 and Foxp3S418E in Stk3/4-deficient Treg cells restores their regulatory functions. (A) Flow cytometric analysis and scatter plot rep- resentation of MFI of p65, CD25, and Foxp3 in Foxp3YFPCre and Foxp3YFPCreStk3Δ/ΔStk4Δ/Δ Treg cells transfected with either empty vector or with p65-encoding retrovirus as indicated (n = 5 per group). (B) Survival of Foxp3ΔEGFPiCre mice injected with empty vector–transfected (n = 8), p65-transfected (n = 5), or Foxp3S418E-transfected Foxp3YFPCreStk3Δ/ΔStk4Δ/Δ Treg cells (n = 5). (C) Flow cytometric analysis and frequencies of CD62LloCD44hi CD4+Foxp3−and CD62LhiCD44lo CD4+Foxp3−Teff cells from spleens of Foxp3ΔEGFPiCre mice injected with Foxp3YFPCreStk3Δ/ΔStk4Δ/Δ Treg cells that had been transfected with empty vector (n = 7), p65 (n = 7), Foxp3S418A

Article Snippet: MycMST1 (#8847) (50), Flag-p65 (#20012) (51), pCL-Eco (#12371) (52), MSCV-IRES-Thy1.1 DEST (#17442), and MSCV-FOXP3IRES-Thy1.1 retroviral vector (#17443) (53) were from Addgene.

Techniques: Over Expression, Transfection, Plasmid Preparation, Injection

Journal: Science immunology

Article Title: A Stk4-Foxp3-NF-κB p65 transcriptional complex promotes T reg cell activation and homeostasis.

doi: 10.1126/sciimmunol.abl8357

Figure Lengend Snippet: Fig. 7. Immune regulatory abnormalities in STK4-deficient patients. (A) Schematic representation of STK4 illustrating its encoding exons, the protein domains, and mapped mutations of four patients. The kinase domain, the inhibitory domain, and the SARAH (Sav/Rassf/Hpo) domain are indicated. (B and C) Flow cytometric analysis and cell frequencies of CD45RA and CCR7 expression on circulating Treg cells (B) and Teff cells (C) of control subjects (n = 4) and STK4-deficient patients (n = 5). (D) Cell frequencies of annexin V and Ki67 expression on circulating Treg and Teff cells of control subjects (n = 4) and STK4-deficient patients (n = 5 for annexin V and n = 4 for Ki67). (E) Cell frequencies and MFI of CD69 and CD25 expression on circulating Treg cells of control and STK4-deficient patients (n = 4 per group). (F) Flow cytometric analysis and MFI of p65 expression on circulating Treg cells of control and STK4-deficient patients (n = 4 per group). (G) Representative histograms of MFI of p-S418 Foxp3 in circulating Treg cells of healthy control (HC) and STK4-deficient patients that were either unstimulated (US) or stimulated with anti-CD3 + IL-2 (n = 4 per group). Each dot represents one patient. Error bars indicate SEM. Statistical tests: Student’s two-tailed t test (B to F) or one-way ANOVA with posttest analysis (G). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Article Snippet: MycMST1 (#8847) (50), Flag-p65 (#20012) (51), pCL-Eco (#12371) (52), MSCV-IRES-Thy1.1 DEST (#17442), and MSCV-FOXP3IRES-Thy1.1 retroviral vector (#17443) (53) were from Addgene.

Techniques: Expressing, Control, Two Tailed Test