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: Development (Cambridge, England)

Article Title: Zinc deficiency causes neural tube defects through attenuation of p53 ubiquitylation

doi: 10.1242/dev.169797

Figure Lengend Snippet: Zinc deficiency attenuates p53 ubiquitylation. (A) Schematic of the regulation of p53 ubiquitylation by the zinc-regulated E3 ubiquitin ligase Mdm2 and by the p53-deubiquitylating enzyme Hausp, leading to p53 degradation or stabilization. The N terminus of Mdm2 binds p53 via a TAZ domain, whereas the C-terminal zinc-regulated RING domain is required for ubiquitin ligase activity towards p53. TAZ, transactive zone. (B) E9.5 primary neuroepithelial cells treated with MG132 proteasome inhibitor and TPEN or TPEN+ZnSO4, followed by p53 co-immunoprecipitation and western blot analysis with antibodies against ubiquitin and Hausp shows TPEN decreases p53 ubiquitylation and Hausp binding. Graph shows western blot quantification data. Immunoprecipitated p53 is used as a loading control. Data are mean±s.d. All treatments were normalized to control. *P<0.05, **P<0.01 by one-way ANOVA analysis. All data came from three independent experiments. (C,D) Typical western blot images (C) and quantification (D) of the different forms of Mdm2 and of Hausp following p53 co-immunprecipitation of brain tissue from E9.5 embryos treated for 3 h in control media or in media containing 10 µM TPEN or 10 µM TPEN+25 µM ZnSO4. In the early embryonic brain we detected full-length Mdm2 (∼130 kDa and ∼90 kDa) and cleaved Mdm2 in which the C terminus is removed (∼55 kDa). All Mdm2 protein forms are bound by p53 in the presence or absence of zinc, but p53 shows reduced binding to full-length Mdm2 isoforms. Hausp binding to p53 is much decreased upon TPEN treatment, despite p53 stabilization (input lane). Immunoprecipitated p53 is used as a loading control. Data are mean±s.d. All treatments were normalized to control. *P<0.05, **P<0.01 and ***P<0.0001 by one-way ANOVA analysis. Each sample was pooled from four or five embryos, all data arise from three independent experiments. (E) Typical gel images of E9.5 primary neuroepithelial cells cultured for 30 min or 2 h in control media or TPEN, followed by p53 co-immunoprecipitation and western blot analysis for Mdm2. Western blot results were quantified (graph). Immunoprecipitated p53 is used as a loading control. Data are mean±s.d. All treatments were normalized to control. *P<0.05 and **P<0.01 by one-way ANOVA analysis. All data arise from two independent experiments. (F,G) CHIP, a zinc-independent E3 ubiquitin ligase of p53, was overexpressed in E9.5 neuroepithelial cells, followed by TPEN treatment for 6 h. CHIP overexpression reduced the number of cleaved Casp3-positive cells, as determined by immunofluorescent staining (F) and quantified in G. *P<0.05 by one-way ANOVA analysis. Data are mean±s.d. All data arise from three independent experiments.

Article Snippet: Plasmids containing the coding region of human p53 ( TP53 -Myc-DDK-tagged) (RC200003, Origene) or human CHIP protein ( STUB1 -Myc-DDK-tagged) (RC200310, Origene) were transfected into cells using Xfect Transfection Reagent (Clontech) according to the manufacturer's protocol (1.5 μg or 2.5 μg plasmid for transfection per well of a 12-well plate; 0.5 or 1 μg for 24-well plate).

Techniques: Ubiquitin Proteomics, Activity Assay, Immunoprecipitation, Western Blot, Binding Assay, Control, Cell Culture, Over Expression, Staining

Journal: Journal of cellular and molecular medicine

Article Title: Aryl Hydrocarbon Receptor Alleviates Hepatic Fibrosis by Inducing Hepatic Stellate Cell Ferroptosis.

doi: 10.1111/jcmm.70278

Figure Lengend Snippet: FIGURE 1 | AHR directly regulates Mrp1 transcription in mHSCs. (A–C) The expression of Ahr, Mrp1 or Cyp1a1 was detected by QPCR. (D) There are two potential AHR exogenous response elements on the Mrp1 promoter sequence. (E) Promoter sequences containing XREL1 and XREL2 were cloned onto PGL3 plasmids, and mutant plasmids were constructed. (F) Double luciferase assay to detect luciferase activity. (G) EMSA detects AHR binding to specific elements XREL1 and XREL2. (H) CHIP detects AHR binding to specific elements XREL1 and XREL2. Data are expressed as means ± SD; *p < 0.05, **p < 0.01 and ***p < 0.001; Student's t-test or one-way ANOVA.

Article Snippet: ChIP assays were performed using the ChIP Assay kit (Beyotime, P2078). mHSCs were treated with YH439 (5 μM) for 24 h. Subsequently, mHSCs were sonicated and then immunoprecipitated with the antibody against AHR (1:100 dilution BOSTER Cat# A00225- 4, RRID: AB_3095576) with IgG (1:100 dilution Proteintech Cat# 30000- 0- AP RRID AB_2819035) as a negative control.

Techniques: Expressing, Sequencing, Clone Assay, Mutagenesis, Construct, Luciferase, Activity Assay, Binding Assay