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: iScience

Article Title: STUB1-mediated ubiquitination regulates the stability of GLUD1 in lung adenocarcinoma

doi: 10.1016/j.isci.2023.107151

Figure Lengend Snippet: STUB1 interacts with GLUD1 and affects the stability of GLUD1 protein (A) Ubibrowser was used to search for E3 ligases of GLUD1. (B and C) In A549 cells, STUB1 was overexpressed (B) or knocked down (C) to detect GLUD1 protein by Western blot. (D) The protein level of GLUD1 and STUB1 in PC9, A549, H292, H358, and H1299 cells were detected by Western blot. (E and F) 293T cells were transfected with pcDNA3.1-His-GLUD1 plasmid (E) or pCMV-Flag-STUB1 (F). The immunoprecipitants were blotted with anti-His or anti-Flag antibodies. WCL: the whole cell lysate. (G and H) In A549 cells, endogenous interaction between GLUD1 and STUB1 was tested using anti-STUB1 (G) and anti-GLUD1 (H) antibodies for co-IP, followed by Western blot detection. (I and J) A549 cells were transfected with Flag-STUB1 plasmid (I) or STUB1 siRNAs (J), the mRNA expression of GLUD1 was detected by RT-PCR. Data represent the average of three independent experiments (mean ± SD). ns, p > 0.05 ∗∗∗p < 0.001. (K and L) A549 cells were transfected with Flag-STUB1 (K) or STUB1 siRNAs (L), cells were treated with CHX (20 μg/mL) for 0, 3, 6, and 12 h. The protein stability of GLUD1 was detected by Western blot (left panel). Relative GLUD1 expression over β-actin was quantified (right panel). Data represent the average of three independent experiments (mean ± SD). ns, p > 0.05; ∗∗p < 0.01 ∗∗∗p < 0.001. (M) In A549 cells, Flag-STUB1 and His-GLUD1 plasmids were co-transfected and treated with MG132 for 12 h. The ubiquitination of GLUD1 was detected. (N and O) In 293T cells, Flag-STUB1 and His-GLUD1 plasmids were co-transfected and treated with MG132 or CQ for 12 h. The K48-linkage ubiquitination (N) and K63-linkage ubiquitination (O) of GLUD1 was detected.

Article Snippet: Anti-STUB1 Recombinant Rabbit Monoclonal Antibody , HUABIO , Cat No: ET7108-65.

Techniques: Western Blot, Transfection, Plasmid Preparation, Co-Immunoprecipitation Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Ubiquitin Proteomics

Journal: iScience

Article Title: STUB1-mediated ubiquitination regulates the stability of GLUD1 in lung adenocarcinoma

doi: 10.1016/j.isci.2023.107151

Figure Lengend Snippet: Lysine 503 of GLUD1 is the key ubiquitination site regulated by STUB1 (A) The ubiquitination sites of GLUD1 predicted by PhosphoSitePlus. (B–M) The GLUD1 lysine residue mutation plasmids (lysine to arginine) are constructed based on the predicted ubiquitin sites of GLUD1. A549 cells were transfected with these plasmids and pCMV-Flag-STUB1, the ubiquitination levels of GLUD1 were detected. (B) GLUD1 K503R (C) GLUD1 K84R (D) GLUD1 K183R (E) GLUD1 K187R (F) GLUD1 K191R (G) GLUD1 K200R (H) GLUD1 K363R (I) GLUD1 K365R (J) GLUD1 K386R (K) GLUD1 K399R (L) GLUD1 K480R (M). In A549 cells, wild-type GLUD1 (His-GLUD1 WT ) or His-GLUD1 K503R plasmids were overexpressed, CHX (20 μg/mL) was added to cells for 0, 3, 6, 9, 12, and 24 h, and the degradation rate of GLUD1 was detected by Western blot (left panel). Relative GLUD1 expression over β-actin was quantified. Data represent the average of three independent experiments (mean ± SD). ∗∗∗p < 0.001 (right panel). (N) A549 cells were transfected with His-GLUD1 K503R alone or co-transfected with Flag-STUB1, cells were treated with CHX (20 μg/mL) for 0, 3, 6, 9, 12, and 24 h. The degradation rate of GLUD1 was detected by Western blot (left panel). Relative GLUD1 expression over β-actin was quantified. Data represent the average of three independent experiments (mean ± SD). ns, p > 0.05 (right panel).

Article Snippet: Anti-STUB1 Recombinant Rabbit Monoclonal Antibody , HUABIO , Cat No: ET7108-65.

Techniques: Ubiquitin Proteomics, Residue, Mutagenesis, Construct, Transfection, Western Blot, Expressing

Journal: iScience

Article Title: STUB1-mediated ubiquitination regulates the stability of GLUD1 in lung adenocarcinoma

doi: 10.1016/j.isci.2023.107151

Figure Lengend Snippet: The regulation of GLUD1 on cell viability relies on STUB1-mediated ubiquitination and degradation (A and B) A549 and H1299 cells were transfected with His-GLUD1 and Flag-STUB1 plasmid or control plasmid. Cell proliferation assays (upper panel) were performed. GLUD1 expression was detected by Western blot (bottom panel). Data represent the average of three independent experiments (mean ± SD). ns, p > 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. (C and D) A549 and H1299 cells were transfected with His-GLUD1 and STUB1 siRNAs. Cell proliferation assay was performed (upper panel). GLUD1 expression was detected by Western blot (bottom panel). Data represent the average of three independent experiments (mean ± SD). ∗p < 0.05, ∗∗p < 0.01. (E) Cell proliferation assay was performed in A549, A549-GLUD1, and A549-GLUD1 K503R cells (left panel). Protein expression was detected by Western blot (right panel). Data represent the average of three independent experiments (mean ± SD). ∗∗∗p < 0.001. (F) Colony formation assay were performed in A549, A549-GLUD1, and A549-GLUD1 K503R cells (left panel). The graph is statistical analysis (right panel). Data represent the average of three independent experiments (mean ± SD). ∗p < 0.05, ∗∗p < 0.01. (G and H) A549-WT, A549-GLUD1, and A549-GLUD1 K503R cells were subcutaneously injected into the flanks of nude mice. After 25 days, tumors were dissected out and photographed (left panel). Tumor weight and volume were measured (right panel). The p value was calculated by paired t test. ∗p < 0.05, ∗∗p < 0.01. (I and J) Immunohistochemical staining of tumors derived from nude mice using anti-Ki67 (I) or anti-TTF1 (J) antibodies. Magnification is ×200.

Article Snippet: Anti-STUB1 Recombinant Rabbit Monoclonal Antibody , HUABIO , Cat No: ET7108-65.

Techniques: Ubiquitin Proteomics, Transfection, Plasmid Preparation, Control, Expressing, Western Blot, Proliferation Assay, Colony Assay, Injection, Immunohistochemical staining, Staining, Derivative Assay

Journal: iScience

Article Title: STUB1-mediated ubiquitination regulates the stability of GLUD1 in lung adenocarcinoma

doi: 10.1016/j.isci.2023.107151

Figure Lengend Snippet:

Article Snippet: Anti-STUB1 Recombinant Rabbit Monoclonal Antibody , HUABIO , Cat No: ET7108-65.

Techniques: Ubiquitin Proteomics, Recombinant, Virus, Transfection, SYBR Green Assay, Construct, Plasmid Preparation, Software

Journal: Scientific Reports

Article Title: Serum N -glycan profiling is a potential biomarker for castration-resistant prostate cancer

doi: 10.1038/s41598-019-53384-y

Figure Lengend Snippet: Detection of lectin-reactive glycans on α-1-acid glycoprotein (AGP) using recombinant lectin array chip. The AGP concentration adjusted lectin array analysis showed that terminal α2.3 sialylated glycan ( A ), α2.6 sialylated glycan ( B ), and terminal galactose ( C ), were significantly increased in the CRPC patients. In contrast, branched-LacNAc structure was significantly decreased in the CRPC patients ( D ). RFI: Relative fluorescent intensity.

Article Snippet: Diluted serum was added to the well of recombinant lectin array chip (Rexxam Co. Ltd., Osaka, Japan).

Techniques: Recombinant, Concentration Assay, Glycoproteomics