Journal: Nature Communications

Article Title: CHIP phosphorylation by protein kinase G enhances protein quality control and attenuates cardiac ischemic injury

doi: 10.1038/s41467-020-18980-x

Figure Lengend Snippet: a Immunoprecipitation of Myc-tagged CHIP with PKG1α. Lanes: 1-input, 2 negative control, 3-IP signal after three bead washes. Experiment replicated ×3. b Mass spectroscopy detects increased Chip-S20 (mouse sequence) phosphorylation with acute PKG activation. The lower schematic shows the sequence and fragmentation matching the spectra (above). The M2S spectra has red annotations for the assigned ion fragments, with three fragment ions localizing phosphorylation at S20 (b8 and y11 indicate it occurs on the N-terminal sequence of LGTGGGGS, b7 indicates the sequence LGTGGGG does not contain the site. Study performed with 3 biological replicates. c CHIP peptide sequence from start of N-terminus shows high level of conservation including three serines (S19, S23, S25 for human; S20, S24, S26 for mouse and rat). Lysine 30 (K30 human, K31 mouse) is also nearby, highly conserved, and known to be critical for CHIP-chaperone binding. d Mass Spec detection of serine phosphorylation of recombinant human CHIP peptide fragment containing residues 13–30 (Uniprot Q9UNE7). Only S20 phosphorylation is detected with WT CHIP. If S20A mutation is expressed, some reduced phosphorylation is observed at S24 and S26. All biological replicates shown in figure, truncated violin plot, median and 25/75%; unpaired t -test, * p = 0.01 vs CHIP-WT. e Recombinant PKG1α/CHIP assay shows pS20 signal by immunoblot for full length WT but not S20A mutant. Example of three biological replicates. f Phospho S20 antibody detects CHIP phosphorylation at S20 in rat myocytes exposed to cGMP or PDE5A inhibition, and both are blocked by concomitant PKG inhibition with DT3 (1 μM). Gel repeated ×3, with 3–4 biological replicates. Summary analysis in Supplementary Fig. .

Article Snippet: Aliquots of recombinant CHIP or PKG1α were diluted to either 12.5 μM or 0.25 μM in Kinase Dilution Buffer II (K23-09, SignalChem: 5 mM MOPS, pH 7.2, 2.5 mM β-glycerol-phosphate, 5 mM MgCl2, 1 mM EGTA, 0.4 mM EDTA, 50 ng/μl BSA, 0.5 mM DTT).

Techniques: Immunoprecipitation, Negative Control, Mass Spectrometry, Sequencing, Phospho-proteomics, Activation Assay, Binding Assay, Recombinant, Mutagenesis, Western Blot, Inhibition

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 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

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

Article Title: E3 Ubiquitin Ligases Cbl-b Regulates Thymic- Derived CD4 + CD25 + Regulatory T Cell Development by Targeting Foxp3 for Ubiquitination

doi: 10.4049/jimmunol.1402434

Figure Lengend Snippet: Cbl-b inducibly associates with Foxp3. (A) BALB/c T cells were stimulated with anti-CD3 and anti-CD28 for 15 min or for 15 and 30 min, and lysed in 0.5% NP-40 lysis buffer. The cell lysates were immunoprecipitated with anti-Foxp3, and blotted with anti-Cbl-b (upper panel) or anti-Stub1 (lower panel). The cell lysates from the unstimulated sample were used as a positive control. The cell lysates immunoprecipitated with rabbit IgG (IgG) were used as a negative control. (B) Schematic design of Cbl-b mutant constructs. (C) HEK293T cells were transfected with HA-tagged Cbl-b, Cbl-b N1/3, Cbl-b C2/3, Cbl-b ΔUBA, together with Flag-tagged Foxp3, and lysed. The cell lysates were immunoprecipitated with anti-Flag, and blotted with anti-HA. (D) Naïve CD4+CD25+ T cells from BALB/c mice were nucleofected with siRNA specific for Stub1, stimulated with anti-CD3 and anti-CD28, and lysed. The cell lysates were immunoprecipitated with anti-Foxp3 and blotted with anti-Cbl-b. The data shown are one representative of two independent experiments.

Article Snippet: GFP-tagged Stub1 plasmid was obtained from OriGene (Rockville, MD).

Techniques: Lysis, Immunoprecipitation, Positive Control, Negative Control, Mutagenesis, Construct, Transfection

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

Article Title: E3 Ubiquitin Ligases Cbl-b Regulates Thymic- Derived CD4 + CD25 + Regulatory T Cell Development by Targeting Foxp3 for Ubiquitination

doi: 10.4049/jimmunol.1402434

Figure Lengend Snippet: Stub1 and Cbl-b sequentially ubiquitinates Foxp3 upon TCR/CD28 stimulation. (A) CD4+CD25+ T cells from WT mice were stimulated with anti-CD3 and anti-CD28, or left unstimulated, and lysed in RIPA buffer. The cell lysates were immunoprecipitated with anti-Foxp3, and blotted with anti-ubiquitin. (B) HEK293T cells were transfected with HA-tagged Cbl-b or Cbl-b C373A, Flag-tagged Foxp3, and His-tagged ubiquitin. The Cell lysates were immunoprecipitated with anti-Flag, and blotted with anti-HA. (C and D) CD4+CD25+ T cells from WT, Cblb−/− or CblbC373A mice, and stimulated with anti-CD3 and anti-CD28, and lysed. The Foxp3 ubiquitination was determined. (E) Cytoplasmic and nuclear fractions of WT CD4+ T cells stimulated with anti-CD3 and anti-CD28 were separated, and immunoprecipitated with anti-Foxp3, and blotted with anti-ubiquitin. (F) WT CD4+ T cells were stimulated with anti-CD3 and anti-CD28 for 1, 2, and 4 hrs in the presence or absence of MG-132. Foxp3 expression was determined. (G) BALB/c CD4+CD25+ T cells were transfected with siRNAs specific for Stub1, Cbl-b, or a scrambled siRNA, and stimulated with anti-CD3 and anti-CD28, and lysed in RIPA buffer. The cell lysates were immunoprecitated with anti-Foxp3, and blotted with anti-ubiquitin. The fold changes of Foxp3 ubiquitination bands in arbitrary densitometric units were determined by the ImageJ 1.48. The data shown are one representative of three independent experiments.

Article Snippet: GFP-tagged Stub1 plasmid was obtained from OriGene (Rockville, MD).

Techniques: Immunoprecipitation, Transfection, Expressing