Journal: Clinical and Experimental Immunology

Article Title: E3 ubiquitin ligases STUB1/CHIP contributes to the Th17/Treg imbalance via the ubiquitination of aryl hydrocarbon receptor in rheumatoid arthritis

doi: 10.1093/cei/uxac072

Figure Lengend Snippet: : The STUB1 protein level in patients with rheumatoid arthritis (RA) and control groups. (A) CD4+ T cells were isolated from RA patients ( n = 4) and healthy controls ( n = 4). The expressive protein levels of STUB1 were performed by Western blot. The STUB1 protein levels were quantified by band intensity and normalized to β-actin levels. STUB1 protein expression in CD4+IL-17+ T (Th17) cells (B) and CD4+Foxp3+ T (Treg) cells (C) from RA PB ( n = 15) and HCs PB( n = 15). (D-F) STUB1 levels in the SF of RA patients were detected, and osteoarthritis patients (OA) were included as a control group. The expression of STUB1 in Th17 cells (D) and Treg cells (E) from RA SF ( n = 8) and controls (OA) SF( n = 8). (F) The expression of STUB1 in Th1 cells from SF of RA patients ( n = 8) and controls (OA) ( n = 8). (G) CD4+ T cells were stimulated with or without TNF-α and IL-6, respectively. The levels of STUB1 were performed by Western blot and data are representative of three independent experiments. ** P <.01 and *** P <.001 vs. healthy controls (Student’s t test). Error bars show mean ± SEM. STUB1, STIP1-homologous U-Box containing protein 1.

Article Snippet: For the detection of STUB1 level in Th1, Th17, or Treg cells, STUB1 antibody (JG38-22) was purchased from Novus Biologicals.

Techniques: Control, Isolation, Western Blot, Expressing

Journal: Clinical and Experimental Immunology

Article Title: E3 ubiquitin ligases STUB1/CHIP contributes to the Th17/Treg imbalance via the ubiquitination of aryl hydrocarbon receptor in rheumatoid arthritis

doi: 10.1093/cei/uxac072

Figure Lengend Snippet: STUB1 affectes Th17 and Treg cell polarization from naive CD4+ T cell. Transfected the lentivirus-expressing STUB1 (LV-STUB1) and LV-sh-STUB1 in isolated CD4+ T cell, stimulated with plate-bound anti-CD3 (5 mg/mL) and anti-CD28 (2 mg/mL) mAbs, and cultured under specific conditions for 5 days. (A) The expression of RORγt, IL-17A and Foxp3 mRNA was evaluated by qRT-PCR in control, LV-STUB1–transfected and LV-sh-STUB1-transfected cells. (B-F) The concentration of IL-17A, IL-6, TNF-α, IL-10 and TGF-β in cell supernatant was detected by ELISA. (G , H) Transfected CD4+ T cells were stimulated with anti-CD3 (5 mg/mL) and anti-CD28 (2 mg/mL) mAbs with Th17 and Treg-polarizing condition, respectively. The proportion of Th17 (CD4+IL-17+) and Treg (CD25+Foxp3+) cells was detected by flow cytometry. Percentages of Th17 cells and Treg cells are shown in the bar. ** P <.01 vs. control groups (Student’s t test). Data are representative of three independent experiments. Error bars show mean ± SEM. IL, interleukin; TNF-α, tumor necrosis factor-α; TGF-β, transforming growth factor-β; qRT-PCR, real-time reverse transcription-polymerase chain reaction.

Article Snippet: For the detection of STUB1 level in Th1, Th17, or Treg cells, STUB1 antibody (JG38-22) was purchased from Novus Biologicals.

Techniques: Transfection, Expressing, Isolation, Cell Culture, Quantitative RT-PCR, Control, Concentration Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Reverse Transcription, Polymerase Chain Reaction

Journal: Clinical and Experimental Immunology

Article Title: E3 ubiquitin ligases STUB1/CHIP contributes to the Th17/Treg imbalance via the ubiquitination of aryl hydrocarbon receptor in rheumatoid arthritis

doi: 10.1093/cei/uxac072

Figure Lengend Snippet: STUB1 physically associates with AHR and promotes the ubiquitination of AHR. (A) Interaction between STUB1 and AHR in the lysates of CD4+ T cells that had been transfected with the indicated plasmids and then were stimulated without or with anti-CD3/anti-CD8. Samples were subjected to immunoprecipitation (IP) with anti-Flag antibody. The immunoprecipitates were immunoblotted (IB) with indicated antibody. (B) Flag-AHR and HA-Ub were co-transfected with different amounts of Myc-STUB1 into HEK293T cells. Polyubiquitination of AHR was detected with the indicated antibody. Cell extracts were immunoblotted with antibody to Flag or Myc tag. β-actin served as a loading control. The AHR protein levels are shown in the bar. (C) Ubiquitination assay for AHR in Jurkat T cells cotransfected with or without STUB1 siRNA and the indicated plasmids. Cell lysates were immunoprecipitated with antibody against Flag and the complex were immunoblotted with antibody to Ubiquitin. Immunoblotting with the indicated antibodies was used to detect the protein level of AHR, STUB1, or β-actin in cell lysates. The AHR protein levels are shown in the bar. (D) HEK293T cells were transfected with Myc-STUB1 and Flag-AHR together with plasmid encoding His-Ub (WT) or the ubiquitin mutants K48R or K63R. The cells were lysed for Co-IP as indicated. The ubiquitination levels are shown in the bar. Data are representative of three independent experiments.

Article Snippet: For the detection of STUB1 level in Th1, Th17, or Treg cells, STUB1 antibody (JG38-22) was purchased from Novus Biologicals.

Techniques: Ubiquitin Proteomics, Transfection, Immunoprecipitation, Control, Western Blot, Plasmid Preparation, Co-Immunoprecipitation Assay

Journal: Clinical and Experimental Immunology

Article Title: E3 ubiquitin ligases STUB1/CHIP contributes to the Th17/Treg imbalance via the ubiquitination of aryl hydrocarbon receptor in rheumatoid arthritis

doi: 10.1093/cei/uxac072

Figure Lengend Snippet: : STUB1 improves the imbalance of Th17/Treg cells in AHR-dependent manner. (A) Ubiquitination of AHR was increased in RA patients compared with healthy controls. Purified CD4+T cells from peripheral blood of RA patients ( n = 4) and healthy controls ( n = 4). AHR ubiquitination was detected with the indicated antibody. Densitometry was performed and quantitation of ubiquitinated AHR was normalized to total AHR from lysates. (B , C) Compared effect of STUB1 on Th17/Treg cells with that of FICZ. The proportion of Th17 (CD4+IL-17+) and Treg (CD25+Foxp3+) cells was detected by flow cytometry. Percentages of Th17 cells and Treg cells are shown in the bar. ** P < .01 vs. control groups. NS, no significant (Student’s t -test). Data are pooled from three independent experiments. Error bars show mean ± SEM.

Article Snippet: For the detection of STUB1 level in Th1, Th17, or Treg cells, STUB1 antibody (JG38-22) was purchased from Novus Biologicals.

Techniques: Ubiquitin Proteomics, Purification, Quantitation Assay, Flow Cytometry, Control

Journal: Clinical and Experimental Immunology

Article Title: E3 ubiquitin ligases STUB1/CHIP contributes to the Th17/Treg imbalance via the ubiquitination of aryl hydrocarbon receptor in rheumatoid arthritis

doi: 10.1093/cei/uxac072

Figure Lengend Snippet: : AHR pathway involves in STUB1-mediated Th17/Treg cell imbalance. CD4+ T cells overexpressing STUB1 were transfected with siAHR or control siRNA and cultured under Th17 or Treg cells polarizing-conditions with anti-CD3/CD28 antibodies treatment. (A) RORγt, IL-17A and Foxp3 gene expression levels were determined by RT-qPCR. (B-F) The concentration of IL-17A, IL-6, TNF-α, IL-10 and TGF-β in supernatant was detected by ELISA. (G , H) The proportion of Th17 (CD4+IL-17+) cells and Treg (CD25+Foxp3+) cells was detected by flow cytometry. Percentages of Th17 cells and Treg cells are shown in the bar. (I, J) The mRNA levels and enzymatic activity of CYP1A1 were evaluated by qRT-PCR and EROD, respectively. ** P < .01 vs. control groups (Student’s t -test). Data are representative of three independent experiments. Error bars show mean ± SEM.

Article Snippet: For the detection of STUB1 level in Th1, Th17, or Treg cells, STUB1 antibody (JG38-22) was purchased from Novus Biologicals.

Techniques: Transfection, Control, Cell Culture, Gene Expression, Quantitative RT-PCR, Concentration Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Activity Assay

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

Article Title: RREB-1 is a transcriptional repressor of HLA-G.

doi: 10.4049/jimmunol.0902053

Figure Lengend Snippet: FIGURE 8. In situ binding of RREB-1 or HDAC1 to the HLA-G pro- moter in a repressive and active-type chromatin. A and B, ChIP performed with JEG-3 (HLA-G ) and M8 (HLA-G ) cells using anti-RREB-1 and anti-HDAC1 Abs on distal and proximal promoter regions (A) Abs target- ing RNA polymerase II (RNApolII), acetylated histone H3 (AcH3), and phosphorylated histone H3 (AcH3 P) on proximal promoter region (B). Immunoprecipitated HLA-G promoter regions are analyzed on agarose gels by semiquantitative HLA-G-specific PCRs targeting proximal and distal HLA-G promoter. Input chromatin (Input) used as PCR control and IgG () are shown. The absence of RREB-1 and HDAC1 binding observed in JEG-3 cells and the absence of RNA polymerase II, acetylated histone H3, and phosphorylated histone H3 binding in M8 cells validate the specificity of Abs used in ChIP assays.

Article Snippet: ChIP assays were performed as previously described (51) using antiRREB-1 from Rockland; anti-acetylated histone H3 (06-599) and antiphosphorylated Ser10 histone H3 (07-081) from Upstate Biotechnology Associates; and anti-RNApolII (C-21) and anti-HDAC1 (H-51) from Santa Cruz Biotechnology.

Techniques: In Situ, Binding Assay, Immunoprecipitation, Control