recombinant chip protein  (MedChemExpress)

Journal: Frontiers in Immunology

Article Title: Targeting the BAG2/CHIP axis promotes gastric cancer apoptosis by blocking apoptosome assembly

doi: 10.3389/fimmu.2025.1578416

Figure Lengend Snippet: FIIN-2 blocks the BAG2-CHIP interaction. (A) Interactions between CHIP and different BAG2 deletion mutants analyzed via co-immunoprecipitation assays. WT, wild type; IP, immunoprecipitation; WCL, whole-cell-lysates. (B) Interactions between BAG2 and different CHIP deletion mutants were examined using co-immunoprecipitation assays. (C) The bound conformation of BAG2 and CHIP as predicted by the Cluspro algorithm. BAG2 is displayed in yellow, and CHIP is displayed in green. (D) This schematic diagram shows the amino acids that interact between BAG2 and CHIP. On the binding surface, the CHIP residue bonds are highlighted in green, while those of BAG2 are in yellow. (E) Interactions between BAG2 and different CHIP deletion mutants containing residues on the binding surface of the mode were analyzed via co-immunoprecipitation assays. (F) Flow diagram of BAG2-CHIP complex inhibitor screening. (G) Computational modeling showcases the interactions between FIIN-2 and CHIP. CHIP is displayed in green, and FIIN-2 is displayed in pink. (H) Microscale thermophoresis (MST) was utilized to ascertain the kinetic constant (Kd) for the interaction between FIIN-2 and CHIP. (I) Co-immunoprecipitation assays of the BAG2-CHIP interaction in cells treated with FIIN-2 at the indicated concentrations in HGC-27. IP, immunoprecipitation; WCL, whole-cell lysates. (J) Western blotting was conducted to assess HSP70 expression levels in cells post-treatment with different FIIN-2 concentrations in HGC-27. (K) An in vitro ubiquitination assay was performed to determine the impact of FIIN-2 (C = 10 μM) on HSP70 ubiquitination, using specified recombinant proteins in HGC-27.

Article Snippet: CHIP human recombinant was purchased from OriGene (cat. TP300310, China).

Techniques: Immunoprecipitation, Binding Assay, Residue, ChIP-chip, Microscale Thermophoresis, Western Blot, Expressing, In Vitro, Ubiquitin Proteomics, Recombinant

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 Cancer Immunology

Article Title: Negative Feedback Expansion of Tregs Caused by Endogenous IL-2 Limits the Activity of IL-2-based Therapies

doi: 10.33696/cancerimmunol.5.074

Figure Lengend Snippet: Figure 1. AU-007 binds to endogenous IL-2 and breaks the negative feedback loop in human PBMCs. A-E: naive hPBMCs were treated once at day 0 with either 1uM AU-007 (red) or with an isotype control antibody (blue). No exogenous IL-2 was added. The culture was monitored for 7 days, and immune cell subpopulations were analyzed daily by flow cytometry. Values were normalized to untreated samples (UNT) at each day. AU-007 completely inhibits Tregs expansion (A) and significantly increases Teffs:Tregs ratio (B), without hindering NKs (C). AU-007 downregulates the suppressive markers of CD4+Treg from panel A, as defined by a significant reduction in mean fluorescence intensity (MFI) of CD25 and FoxP3 (D-E). F-K: Total hPBMCs were stimulated for 24h with anti-CD3/anti-CD28 (stimulation only, green) or stimulated with anti-CD3/anti-CD28 in the presence of 200nM of AU-007 mAb (red) or with 200nM of isotype control mAb (blue). No exogenous IL-2 was added. Immune cells subpopulations were analyzed by flow cytometry. AU-007 inhibits Tregs without hindering effector cells and NKs (F-I). AU-007 downregulates the suppressive markers of CD4+Treg from panel G, as defined by a significant reduction in MFI of CD25 and FoxP3 (J-K).

Article Snippet: epitopes To test whether the CD25 epitope is blocked in the nonalpha-IL-2 format, a biotinylated human CD25 (AVI10305-050, R&D systems) was captured on a streptavidin-coated chip (BR100531, Cytiva) and 200 nM of human IL-2 (RKP60568, Reprokine) or non-alpha-IL-2 (IL-2 conjugated to CD25 subunit) were injected for 60 seconds.

Techniques: Control, Flow Cytometry, Fluorescence

Journal: Journal of Cancer Immunology

Article Title: Negative Feedback Expansion of Tregs Caused by Endogenous IL-2 Limits the Activity of IL-2-based Therapies

doi: 10.33696/cancerimmunol.5.074

Figure Lengend Snippet: Figure 2. AU-007 can capture and redirect endogenous IL-2 to break the auto-inhibitory loop in hPBMCs while HD IL-2 or naIL-2 cannot. AU-007 promotes the expansion of NKs and CD8 T-cells while completely inhibiting the expansion of regulatory T-cells. A-E: naive hPBMCs were treated once on day 0 with 1nM of naIL-2 (purple) or with HD IL-2 (1nM) combined with 1uM of isotype control Ab (black) or with either 1uM AU-007 (red) or 10uM AU-007 (turquoise). The culture was monitored for 7 days, and immune cell subpopulations were analyzed daily by flow cytometry. Values were normalized to untreated samples (UNT) at each day. While naIL-2 expands NKs similarly to AU-007 it fails to inhibit Tregs expansion, while AU-007 completely inhibits Tregs expansion in culture (A) and significantly increases the Teffs:Tregs ratio (B), without hindering NKs (C). AU-007 downregulates the suppressive markers of CD4+Treg from panel A, as defined by a significant reduction in MFI of CD25 and FoxP3 (D-E). F-H: AU-007 rescues activated lymphocyte viability decreased by treatment with HD IL-2. hPBMCs culture was stimulated once with anti-CD3/anti-CD28 Abs with or without 10uM of AU-007. 3 days post-stimulation all samples were given HD IL-2 (1nM) and were monitored daily for cell viability using flow cytometry.

Article Snippet: epitopes To test whether the CD25 epitope is blocked in the nonalpha-IL-2 format, a biotinylated human CD25 (AVI10305-050, R&D systems) was captured on a streptavidin-coated chip (BR100531, Cytiva) and 200 nM of human IL-2 (RKP60568, Reprokine) or non-alpha-IL-2 (IL-2 conjugated to CD25 subunit) were injected for 60 seconds.

Techniques: Control, Flow Cytometry

Journal: Journal of Cancer Immunology

Article Title: Negative Feedback Expansion of Tregs Caused by Endogenous IL-2 Limits the Activity of IL-2-based Therapies

doi: 10.33696/cancerimmunol.5.074

Figure Lengend Snippet: Figure 3. IL-2 negative feedback loop caused by endogenous IL-2 limits the activity of modified IL-2-based therapies. A. Schematic representation of IL-2 role as an immunomodulator in homeostasis and inflammation. B. Exogenous administration of modified IL-2 with bias selectivity to dimer-expressing cells promotes the expansion of CD25 negative (CD25-) effector cells yet is undermined by the endogenous IL-2 that pushes the system back to homeostasis. C. AU-007 captures and redirects endogenous IL-2, allowing it to expand CD25 negative (CD25-) effector cells while breaking the auto-inhibitory loop and expanding the inflammation & immune stimulation stage.

Article Snippet: epitopes To test whether the CD25 epitope is blocked in the nonalpha-IL-2 format, a biotinylated human CD25 (AVI10305-050, R&D systems) was captured on a streptavidin-coated chip (BR100531, Cytiva) and 200 nM of human IL-2 (RKP60568, Reprokine) or non-alpha-IL-2 (IL-2 conjugated to CD25 subunit) were injected for 60 seconds.

Techniques: Activity Assay, Modification, Expressing