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cycloheximide  (MedChemExpress)


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    Structured Review

    MedChemExpress cycloheximide
    A Flowchart of mass spectrometry analysis to identify proteins interacting with TRAF2 in U251 cells. B The interaction between endogenous TRAF2 and FASN was verified in U251 and A172 cells by co-immunoprecipitation (Co-IP) assay. C Co-immunoprecipitation (Co-IP) assay was performed to detect the interaction between TRAF2 and FASN in HEK293T cells co-transfected with His-TRAF2 and Flag-FASN. D IF staining analysis confirmed the cytoplasmic co-localization of TRAF2 and FASN proteins in U251 and A172 cells. Endogenous TRAF2 and FASN were stained with anti-TRAF2 (green) and anti-FASN (red) antibodies, respectively. Scale bar, 20 μm. E Western blot analysis of FASN protein expression in U251 and A172 cells overexpressing TRAF2. F Western blot analysis of FASN protein expression in U251 and A172 cells with TRAF2 knockdown. G FASN protein expression was assessed by Western blot in HEK293T cells transfected with Flag-FASN and increasing doses of His-TRAF2 plasmid. H U251 and A172 cells were transiently transfected to express His-TRAF2. Immunofluorescence analysis was performed using anti-His-TRAF2 (green) and anti-FASN (red) antibodies. Nuclei were stained with Hoechst (blue). White arrows indicate His-TRAF2-positive cells. Scale bar, 30 μm. I Western blot analysis of lysates from U251 and A172 cells with TRAF2 knockdown treated with DMSO, MG132 (10 μM), or chloroquine (25 μM). J RT-qPCR analysis of FASN mRNA expression levels in U251 and A172 cells with TRAF2 overexpression or knockdown ( n = 5, Student’s t -test, ns not significant). K <t>Cycloheximide</t> (CHX, 100 μg/ml) was used to treat U251 and A172 cells overexpressing TRAF2 to block new protein synthesis and measure the protein half-life of FASN. Protein stability was analyzed using ImageJ ( n = 5, Student’s t -test, ** p < 0.01). L Cycloheximide (CHX, 100 μg/ml) was used to treat U251 and A172 cells with TRAF2 knockdown to block new protein synthesis and measure the protein half-life of FASN. Protein stability was analyzed using ImageJ ( n = 5, Student’s t -test, ** p < 0.01).
    Cycloheximide, supplied by MedChemExpress, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/hy-n8183/Shancigusin+I/pmc13187350-394-0-24
    Average 94 stars, based on 1 article reviews
    cycloheximide - by Bioz Stars, 2026-10
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    Images

    1) Product Images from "Low-dose TNF-α drives malignant progression and lipid metabolism in glioblastoma through the TRAF2-FASN axis"

    Article Title: Low-dose TNF-α drives malignant progression and lipid metabolism in glioblastoma through the TRAF2-FASN axis

    Journal: Cell Death Discovery

    doi: 10.1038/s41420-026-03087-x

    A Flowchart of mass spectrometry analysis to identify proteins interacting with TRAF2 in U251 cells. B The interaction between endogenous TRAF2 and FASN was verified in U251 and A172 cells by co-immunoprecipitation (Co-IP) assay. C Co-immunoprecipitation (Co-IP) assay was performed to detect the interaction between TRAF2 and FASN in HEK293T cells co-transfected with His-TRAF2 and Flag-FASN. D IF staining analysis confirmed the cytoplasmic co-localization of TRAF2 and FASN proteins in U251 and A172 cells. Endogenous TRAF2 and FASN were stained with anti-TRAF2 (green) and anti-FASN (red) antibodies, respectively. Scale bar, 20 μm. E Western blot analysis of FASN protein expression in U251 and A172 cells overexpressing TRAF2. F Western blot analysis of FASN protein expression in U251 and A172 cells with TRAF2 knockdown. G FASN protein expression was assessed by Western blot in HEK293T cells transfected with Flag-FASN and increasing doses of His-TRAF2 plasmid. H U251 and A172 cells were transiently transfected to express His-TRAF2. Immunofluorescence analysis was performed using anti-His-TRAF2 (green) and anti-FASN (red) antibodies. Nuclei were stained with Hoechst (blue). White arrows indicate His-TRAF2-positive cells. Scale bar, 30 μm. I Western blot analysis of lysates from U251 and A172 cells with TRAF2 knockdown treated with DMSO, MG132 (10 μM), or chloroquine (25 μM). J RT-qPCR analysis of FASN mRNA expression levels in U251 and A172 cells with TRAF2 overexpression or knockdown ( n = 5, Student’s t -test, ns not significant). K Cycloheximide (CHX, 100 μg/ml) was used to treat U251 and A172 cells overexpressing TRAF2 to block new protein synthesis and measure the protein half-life of FASN. Protein stability was analyzed using ImageJ ( n = 5, Student’s t -test, ** p < 0.01). L Cycloheximide (CHX, 100 μg/ml) was used to treat U251 and A172 cells with TRAF2 knockdown to block new protein synthesis and measure the protein half-life of FASN. Protein stability was analyzed using ImageJ ( n = 5, Student’s t -test, ** p < 0.01).
    Figure Legend Snippet: A Flowchart of mass spectrometry analysis to identify proteins interacting with TRAF2 in U251 cells. B The interaction between endogenous TRAF2 and FASN was verified in U251 and A172 cells by co-immunoprecipitation (Co-IP) assay. C Co-immunoprecipitation (Co-IP) assay was performed to detect the interaction between TRAF2 and FASN in HEK293T cells co-transfected with His-TRAF2 and Flag-FASN. D IF staining analysis confirmed the cytoplasmic co-localization of TRAF2 and FASN proteins in U251 and A172 cells. Endogenous TRAF2 and FASN were stained with anti-TRAF2 (green) and anti-FASN (red) antibodies, respectively. Scale bar, 20 μm. E Western blot analysis of FASN protein expression in U251 and A172 cells overexpressing TRAF2. F Western blot analysis of FASN protein expression in U251 and A172 cells with TRAF2 knockdown. G FASN protein expression was assessed by Western blot in HEK293T cells transfected with Flag-FASN and increasing doses of His-TRAF2 plasmid. H U251 and A172 cells were transiently transfected to express His-TRAF2. Immunofluorescence analysis was performed using anti-His-TRAF2 (green) and anti-FASN (red) antibodies. Nuclei were stained with Hoechst (blue). White arrows indicate His-TRAF2-positive cells. Scale bar, 30 μm. I Western blot analysis of lysates from U251 and A172 cells with TRAF2 knockdown treated with DMSO, MG132 (10 μM), or chloroquine (25 μM). J RT-qPCR analysis of FASN mRNA expression levels in U251 and A172 cells with TRAF2 overexpression or knockdown ( n = 5, Student’s t -test, ns not significant). K Cycloheximide (CHX, 100 μg/ml) was used to treat U251 and A172 cells overexpressing TRAF2 to block new protein synthesis and measure the protein half-life of FASN. Protein stability was analyzed using ImageJ ( n = 5, Student’s t -test, ** p < 0.01). L Cycloheximide (CHX, 100 μg/ml) was used to treat U251 and A172 cells with TRAF2 knockdown to block new protein synthesis and measure the protein half-life of FASN. Protein stability was analyzed using ImageJ ( n = 5, Student’s t -test, ** p < 0.01).

    Techniques Used: Mass Spectrometry, Co-Immunoprecipitation Assay, Transfection, Staining, Western Blot, Expressing, Knockdown, Plasmid Preparation, Immunofluorescence, Quantitative RT-PCR, Over Expression, Blocking Assay

    A U251 and A172 cells were treated with TNF-α (10 ng/mL) for indicated durations, and endogenous FASN and TRAF2 protein levels were analyzed by Western blot. B U251 and A172 cells stimulated with TNF-α (10 ng/mL) were treated with cycloheximide (CHX, 100 μg/mL) to block new protein synthesis. The protein half-life of FASN was measured and protein stability was analyzed using ImageJ ( n = 5, Student’s t -test, ** p < 0.01). C U251 and A172 cells stimulated with TNF-α (10 ng/mL) were treated with cycloheximide (CHX, 100 μg/mL) to block new protein synthesis. The protein half-life of TRAF2 was measured and protein stability was analyzed using ImageJ ( n = 5, Student’s t -test, ns: not significant). D RT-qPCR analysis of FASN mRNA expression levels in U251 and A172 cells treated with TNF-α (10 ng/mL) for 24 h ( n = 5, Student’s t -test, ns: not significant). E Endogenous FASN and TRAF2 protein levels were detected by Western blot in TRAF2-knockdown U251 and A172 cells re-exposed to TNF-α (10 ng/mL) stimulation. F Co-immunoprecipitation analysis of TRAF2 and FASN in U251 and A172 cells after stimulation with TNF-α (10 ng/mL) for indicated durations. G HEK293T cells co-transfected with HIS-TRAF2 and FLAG-FASN plasmids were treated with TNF-α (10 ng/mL) for indicated durations. Co-immunoprecipitation (Co-IP) was performed to assess the effect of TNF-α on the TRAF2-FASN interaction. H , I Levels of total ubiquitination and K63-linked polyubiquitination co-immunoprecipitated with TRAF2 were detected in U251 and A172 cells stimulated with TNF-α (10 ng/mL). J HEK293T cells co-transfected with HIS-TRAF2 and HA-Ub (WT or K63-only) plasmids were treated with or without TNF-α (10 ng/mL) stimulation. Immunoprecipitation was performed using an anti-HIS antibody to detect TRAF2 ubiquitination levels. K , L Levels of total ubiquitination and K63-linked polyubiquitination co-immunoprecipitated with FASN were detected in U251 and A172 cells stimulated with TNF-α (10 ng/mL). M HEK293T cells co-transfected with FLAG-FASN, HA-Ub (WT or K63-only), and HIS-TRAF2 plasmids were treated with or without TNF-α (10 ng/mL) stimulation. Immunoprecipitation was performed using an anti-FLAG antibody to detect FASN ubiquitination levels. N TRAF2 knockdown abolished the TNF-α-induced upregulation of endogenous FASN ubiquitination and protein levels in U251 and A172 cells.
    Figure Legend Snippet: A U251 and A172 cells were treated with TNF-α (10 ng/mL) for indicated durations, and endogenous FASN and TRAF2 protein levels were analyzed by Western blot. B U251 and A172 cells stimulated with TNF-α (10 ng/mL) were treated with cycloheximide (CHX, 100 μg/mL) to block new protein synthesis. The protein half-life of FASN was measured and protein stability was analyzed using ImageJ ( n = 5, Student’s t -test, ** p < 0.01). C U251 and A172 cells stimulated with TNF-α (10 ng/mL) were treated with cycloheximide (CHX, 100 μg/mL) to block new protein synthesis. The protein half-life of TRAF2 was measured and protein stability was analyzed using ImageJ ( n = 5, Student’s t -test, ns: not significant). D RT-qPCR analysis of FASN mRNA expression levels in U251 and A172 cells treated with TNF-α (10 ng/mL) for 24 h ( n = 5, Student’s t -test, ns: not significant). E Endogenous FASN and TRAF2 protein levels were detected by Western blot in TRAF2-knockdown U251 and A172 cells re-exposed to TNF-α (10 ng/mL) stimulation. F Co-immunoprecipitation analysis of TRAF2 and FASN in U251 and A172 cells after stimulation with TNF-α (10 ng/mL) for indicated durations. G HEK293T cells co-transfected with HIS-TRAF2 and FLAG-FASN plasmids were treated with TNF-α (10 ng/mL) for indicated durations. Co-immunoprecipitation (Co-IP) was performed to assess the effect of TNF-α on the TRAF2-FASN interaction. H , I Levels of total ubiquitination and K63-linked polyubiquitination co-immunoprecipitated with TRAF2 were detected in U251 and A172 cells stimulated with TNF-α (10 ng/mL). J HEK293T cells co-transfected with HIS-TRAF2 and HA-Ub (WT or K63-only) plasmids were treated with or without TNF-α (10 ng/mL) stimulation. Immunoprecipitation was performed using an anti-HIS antibody to detect TRAF2 ubiquitination levels. K , L Levels of total ubiquitination and K63-linked polyubiquitination co-immunoprecipitated with FASN were detected in U251 and A172 cells stimulated with TNF-α (10 ng/mL). M HEK293T cells co-transfected with FLAG-FASN, HA-Ub (WT or K63-only), and HIS-TRAF2 plasmids were treated with or without TNF-α (10 ng/mL) stimulation. Immunoprecipitation was performed using an anti-FLAG antibody to detect FASN ubiquitination levels. N TRAF2 knockdown abolished the TNF-α-induced upregulation of endogenous FASN ubiquitination and protein levels in U251 and A172 cells.

    Techniques Used: Western Blot, Blocking Assay, Quantitative RT-PCR, Expressing, Knockdown, Immunoprecipitation, Transfection, Co-Immunoprecipitation Assay, Ubiquitin Proteomics

    A Simulated binding conformations of candidate small-molecule compounds obtained through virtual screening targeting the RING domain essential for TRAF2 catalytic activity. B Endogenous FASN and TRAF2 protein levels were analyzed by Western blot in U251 and A172 cells treated with gradient concentrations of Jionoside B1 for 24 h. C Endogenous FASN and TRAF2 protein levels were detected by Western blot in U251 and A172 cells treated with Jionoside B1 (100 μM) for indicated durations. D U251 and A172 cells stimulated with Jionoside B1 (100 μM) were treated with cycloheximide (CHX, 100 μg/mL) to block new protein synthesis. The protein half-life of FASN was measured and protein stability was analyzed using ImageJ ( n = 5, Student’s t -test, ** p < 0.01). E U251 and A172 cells stimulated with Jionoside B1 (100 μM) were treated with cycloheximide (CHX, 100 μg/mL) to block new protein synthesis. The protein half-life of TRAF2 was measured and protein stability was analyzed using ImageJ ( n = 5, Student’s t -test, ns: not significant). F . Endogenous FASN protein levels were detected by Western blot in TRAF2-overexpressing U251 and A172 cells re-exposed to Jionoside B1 (100 μM) stimulation. G U251 and A172 cells were treated with TNF-α (10 ng/mL) alone or in combination with Jionoside B1 (100 μM) for 24 hours. H , I Levels of total ubiquitination and K63-linked polyubiquitination co-immunoprecipitated with FASN were detected in U251 and A172 cells stimulated with Jionoside B1 (100 μM). J . HEK293T cells co-transfected with FLAG-FASN and HA-Ub (K63-only) plasmids were treated with or without Jionoside B1 (100 μM) stimulation. Immunoprecipitation was performed using an anti-FLAG antibody to detect K63-linked ubiquitination of FASN. K Co-immunoprecipitation analysis of TRAF2 and FASN in U251 and A172 cells after stimulation with Jionoside B1 (100 μM) for indicated durations. L HEK293T cells co-transfected with HIS-TRAF2 and FLAG-FASN plasmids were treated with Jionoside B1 (100 μM) for indicated durations. Co-immunoprecipitation (Co-IP) was performed to assess the effect of Jionoside B1 on the TRAF2-FASN interaction. M Schematic diagram of in vivo evaluation of Jionoside B1’s tumor growth inhibition. Luciferase-expressing U251 cells were implanted into the brains of nude mice, and intraperitoneal injections of DMSO or Jionoside B1 (20 mg/kg/day) were administered every 2 days starting from day 14 post-surgery. Tumor volume was monitored by in vivo imaging on days 14 and 28 post-surgery. N In vivo imaging of orthotopic intracranial tumors. O Statistical results of tumor volumes ( n = 4, Student’s t -test, *** p < 0.001).
    Figure Legend Snippet: A Simulated binding conformations of candidate small-molecule compounds obtained through virtual screening targeting the RING domain essential for TRAF2 catalytic activity. B Endogenous FASN and TRAF2 protein levels were analyzed by Western blot in U251 and A172 cells treated with gradient concentrations of Jionoside B1 for 24 h. C Endogenous FASN and TRAF2 protein levels were detected by Western blot in U251 and A172 cells treated with Jionoside B1 (100 μM) for indicated durations. D U251 and A172 cells stimulated with Jionoside B1 (100 μM) were treated with cycloheximide (CHX, 100 μg/mL) to block new protein synthesis. The protein half-life of FASN was measured and protein stability was analyzed using ImageJ ( n = 5, Student’s t -test, ** p < 0.01). E U251 and A172 cells stimulated with Jionoside B1 (100 μM) were treated with cycloheximide (CHX, 100 μg/mL) to block new protein synthesis. The protein half-life of TRAF2 was measured and protein stability was analyzed using ImageJ ( n = 5, Student’s t -test, ns: not significant). F . Endogenous FASN protein levels were detected by Western blot in TRAF2-overexpressing U251 and A172 cells re-exposed to Jionoside B1 (100 μM) stimulation. G U251 and A172 cells were treated with TNF-α (10 ng/mL) alone or in combination with Jionoside B1 (100 μM) for 24 hours. H , I Levels of total ubiquitination and K63-linked polyubiquitination co-immunoprecipitated with FASN were detected in U251 and A172 cells stimulated with Jionoside B1 (100 μM). J . HEK293T cells co-transfected with FLAG-FASN and HA-Ub (K63-only) plasmids were treated with or without Jionoside B1 (100 μM) stimulation. Immunoprecipitation was performed using an anti-FLAG antibody to detect K63-linked ubiquitination of FASN. K Co-immunoprecipitation analysis of TRAF2 and FASN in U251 and A172 cells after stimulation with Jionoside B1 (100 μM) for indicated durations. L HEK293T cells co-transfected with HIS-TRAF2 and FLAG-FASN plasmids were treated with Jionoside B1 (100 μM) for indicated durations. Co-immunoprecipitation (Co-IP) was performed to assess the effect of Jionoside B1 on the TRAF2-FASN interaction. M Schematic diagram of in vivo evaluation of Jionoside B1’s tumor growth inhibition. Luciferase-expressing U251 cells were implanted into the brains of nude mice, and intraperitoneal injections of DMSO or Jionoside B1 (20 mg/kg/day) were administered every 2 days starting from day 14 post-surgery. Tumor volume was monitored by in vivo imaging on days 14 and 28 post-surgery. N In vivo imaging of orthotopic intracranial tumors. O Statistical results of tumor volumes ( n = 4, Student’s t -test, *** p < 0.001).

    Techniques Used: Binding Assay, Activity Assay, Western Blot, Blocking Assay, Ubiquitin Proteomics, Immunoprecipitation, Transfection, Co-Immunoprecipitation Assay, In Vivo, Inhibition, Luciferase, Expressing, In Vivo Imaging

    Related Articles

    Mass Spectrometry:

    Article Title: Low-dose TNF-α drives malignant progression and lipid metabolism in glioblastoma through the TRAF2-FASN axis
    Article Snippet: The recombinant human TNF-α protein (#16769) was purchased from Cell Signaling Technology.The recombinant human TNF-α protein (#16769) was purchased from Cell Signaling Technology.. Cycloheximide (HY-12320), MG-132 (HY-13259), Chloroquine (HY-17589A), R-7050 (HY-110203), Shancigusin I (HY-N8183), Rebaudioside F (HY-N6887), Jionoside B1 (HY-N2218), and Carnostatine hydrochloride (HY-112431A) were obtained from MedChemExpress.

    Co-Immunoprecipitation Assay:

    Article Title: Low-dose TNF-α drives malignant progression and lipid metabolism in glioblastoma through the TRAF2-FASN axis
    Article Snippet: The recombinant human TNF-α protein (#16769) was purchased from Cell Signaling Technology.The recombinant human TNF-α protein (#16769) was purchased from Cell Signaling Technology.. Cycloheximide (HY-12320), MG-132 (HY-13259), Chloroquine (HY-17589A), R-7050 (HY-110203), Shancigusin I (HY-N8183), Rebaudioside F (HY-N6887), Jionoside B1 (HY-N2218), and Carnostatine hydrochloride (HY-112431A) were obtained from MedChemExpress.

    Transfection:

    Article Title: Low-dose TNF-α drives malignant progression and lipid metabolism in glioblastoma through the TRAF2-FASN axis
    Article Snippet: The recombinant human TNF-α protein (#16769) was purchased from Cell Signaling Technology.The recombinant human TNF-α protein (#16769) was purchased from Cell Signaling Technology.. Cycloheximide (HY-12320), MG-132 (HY-13259), Chloroquine (HY-17589A), R-7050 (HY-110203), Shancigusin I (HY-N8183), Rebaudioside F (HY-N6887), Jionoside B1 (HY-N2218), and Carnostatine hydrochloride (HY-112431A) were obtained from MedChemExpress.

    Staining:

    Article Title: Low-dose TNF-α drives malignant progression and lipid metabolism in glioblastoma through the TRAF2-FASN axis
    Article Snippet: The recombinant human TNF-α protein (#16769) was purchased from Cell Signaling Technology.The recombinant human TNF-α protein (#16769) was purchased from Cell Signaling Technology.. Cycloheximide (HY-12320), MG-132 (HY-13259), Chloroquine (HY-17589A), R-7050 (HY-110203), Shancigusin I (HY-N8183), Rebaudioside F (HY-N6887), Jionoside B1 (HY-N2218), and Carnostatine hydrochloride (HY-112431A) were obtained from MedChemExpress.

    Western Blot:

    Article Title: Low-dose TNF-α drives malignant progression and lipid metabolism in glioblastoma through the TRAF2-FASN axis
    Article Snippet: The recombinant human TNF-α protein (#16769) was purchased from Cell Signaling Technology.The recombinant human TNF-α protein (#16769) was purchased from Cell Signaling Technology.. Cycloheximide (HY-12320), MG-132 (HY-13259), Chloroquine (HY-17589A), R-7050 (HY-110203), Shancigusin I (HY-N8183), Rebaudioside F (HY-N6887), Jionoside B1 (HY-N2218), and Carnostatine hydrochloride (HY-112431A) were obtained from MedChemExpress.

    Expressing:

    Article Title: Low-dose TNF-α drives malignant progression and lipid metabolism in glioblastoma through the TRAF2-FASN axis
    Article Snippet: The recombinant human TNF-α protein (#16769) was purchased from Cell Signaling Technology.The recombinant human TNF-α protein (#16769) was purchased from Cell Signaling Technology.. Cycloheximide (HY-12320), MG-132 (HY-13259), Chloroquine (HY-17589A), R-7050 (HY-110203), Shancigusin I (HY-N8183), Rebaudioside F (HY-N6887), Jionoside B1 (HY-N2218), and Carnostatine hydrochloride (HY-112431A) were obtained from MedChemExpress.

    Knockdown:

    Article Title: Low-dose TNF-α drives malignant progression and lipid metabolism in glioblastoma through the TRAF2-FASN axis
    Article Snippet: The recombinant human TNF-α protein (#16769) was purchased from Cell Signaling Technology.The recombinant human TNF-α protein (#16769) was purchased from Cell Signaling Technology.. Cycloheximide (HY-12320), MG-132 (HY-13259), Chloroquine (HY-17589A), R-7050 (HY-110203), Shancigusin I (HY-N8183), Rebaudioside F (HY-N6887), Jionoside B1 (HY-N2218), and Carnostatine hydrochloride (HY-112431A) were obtained from MedChemExpress.

    Plasmid Preparation:

    Article Title: Low-dose TNF-α drives malignant progression and lipid metabolism in glioblastoma through the TRAF2-FASN axis
    Article Snippet: The recombinant human TNF-α protein (#16769) was purchased from Cell Signaling Technology.The recombinant human TNF-α protein (#16769) was purchased from Cell Signaling Technology.. Cycloheximide (HY-12320), MG-132 (HY-13259), Chloroquine (HY-17589A), R-7050 (HY-110203), Shancigusin I (HY-N8183), Rebaudioside F (HY-N6887), Jionoside B1 (HY-N2218), and Carnostatine hydrochloride (HY-112431A) were obtained from MedChemExpress.

    Immunofluorescence:

    Article Title: Low-dose TNF-α drives malignant progression and lipid metabolism in glioblastoma through the TRAF2-FASN axis
    Article Snippet: The recombinant human TNF-α protein (#16769) was purchased from Cell Signaling Technology.The recombinant human TNF-α protein (#16769) was purchased from Cell Signaling Technology.. Cycloheximide (HY-12320), MG-132 (HY-13259), Chloroquine (HY-17589A), R-7050 (HY-110203), Shancigusin I (HY-N8183), Rebaudioside F (HY-N6887), Jionoside B1 (HY-N2218), and Carnostatine hydrochloride (HY-112431A) were obtained from MedChemExpress.

    Quantitative RT-PCR:

    Article Title: Low-dose TNF-α drives malignant progression and lipid metabolism in glioblastoma through the TRAF2-FASN axis
    Article Snippet: The recombinant human TNF-α protein (#16769) was purchased from Cell Signaling Technology.The recombinant human TNF-α protein (#16769) was purchased from Cell Signaling Technology.. Cycloheximide (HY-12320), MG-132 (HY-13259), Chloroquine (HY-17589A), R-7050 (HY-110203), Shancigusin I (HY-N8183), Rebaudioside F (HY-N6887), Jionoside B1 (HY-N2218), and Carnostatine hydrochloride (HY-112431A) were obtained from MedChemExpress.

    Over Expression:

    Article Title: Low-dose TNF-α drives malignant progression and lipid metabolism in glioblastoma through the TRAF2-FASN axis
    Article Snippet: The recombinant human TNF-α protein (#16769) was purchased from Cell Signaling Technology.The recombinant human TNF-α protein (#16769) was purchased from Cell Signaling Technology.. Cycloheximide (HY-12320), MG-132 (HY-13259), Chloroquine (HY-17589A), R-7050 (HY-110203), Shancigusin I (HY-N8183), Rebaudioside F (HY-N6887), Jionoside B1 (HY-N2218), and Carnostatine hydrochloride (HY-112431A) were obtained from MedChemExpress.

    Blocking Assay:

    Article Title: Low-dose TNF-α drives malignant progression and lipid metabolism in glioblastoma through the TRAF2-FASN axis
    Article Snippet: The recombinant human TNF-α protein (#16769) was purchased from Cell Signaling Technology.The recombinant human TNF-α protein (#16769) was purchased from Cell Signaling Technology.. Cycloheximide (HY-12320), MG-132 (HY-13259), Chloroquine (HY-17589A), R-7050 (HY-110203), Shancigusin I (HY-N8183), Rebaudioside F (HY-N6887), Jionoside B1 (HY-N2218), and Carnostatine hydrochloride (HY-112431A) were obtained from MedChemExpress.

    Immunoprecipitation:

    Article Title: Low-dose TNF-α drives malignant progression and lipid metabolism in glioblastoma through the TRAF2-FASN axis
    Article Snippet: The recombinant human TNF-α protein (#16769) was purchased from Cell Signaling Technology.The recombinant human TNF-α protein (#16769) was purchased from Cell Signaling Technology.. Cycloheximide (HY-12320), MG-132 (HY-13259), Chloroquine (HY-17589A), R-7050 (HY-110203), Shancigusin I (HY-N8183), Rebaudioside F (HY-N6887), Jionoside B1 (HY-N2218), and Carnostatine hydrochloride (HY-112431A) were obtained from MedChemExpress.

    Ubiquitin Proteomics:

    Article Title: Low-dose TNF-α drives malignant progression and lipid metabolism in glioblastoma through the TRAF2-FASN axis
    Article Snippet: The recombinant human TNF-α protein (#16769) was purchased from Cell Signaling Technology.The recombinant human TNF-α protein (#16769) was purchased from Cell Signaling Technology.. Cycloheximide (HY-12320), MG-132 (HY-13259), Chloroquine (HY-17589A), R-7050 (HY-110203), Shancigusin I (HY-N8183), Rebaudioside F (HY-N6887), Jionoside B1 (HY-N2218), and Carnostatine hydrochloride (HY-112431A) were obtained from MedChemExpress.

    Binding Assay:

    Article Title: Low-dose TNF-α drives malignant progression and lipid metabolism in glioblastoma through the TRAF2-FASN axis
    Article Snippet: The recombinant human TNF-α protein (#16769) was purchased from Cell Signaling Technology.The recombinant human TNF-α protein (#16769) was purchased from Cell Signaling Technology.. Cycloheximide (HY-12320), MG-132 (HY-13259), Chloroquine (HY-17589A), R-7050 (HY-110203), Shancigusin I (HY-N8183), Rebaudioside F (HY-N6887), Jionoside B1 (HY-N2218), and Carnostatine hydrochloride (HY-112431A) were obtained from MedChemExpress.

    Activity Assay:

    Article Title: Low-dose TNF-α drives malignant progression and lipid metabolism in glioblastoma through the TRAF2-FASN axis
    Article Snippet: The recombinant human TNF-α protein (#16769) was purchased from Cell Signaling Technology.The recombinant human TNF-α protein (#16769) was purchased from Cell Signaling Technology.. Cycloheximide (HY-12320), MG-132 (HY-13259), Chloroquine (HY-17589A), R-7050 (HY-110203), Shancigusin I (HY-N8183), Rebaudioside F (HY-N6887), Jionoside B1 (HY-N2218), and Carnostatine hydrochloride (HY-112431A) were obtained from MedChemExpress.

    In Vivo:

    Article Title: Low-dose TNF-α drives malignant progression and lipid metabolism in glioblastoma through the TRAF2-FASN axis
    Article Snippet: The recombinant human TNF-α protein (#16769) was purchased from Cell Signaling Technology.The recombinant human TNF-α protein (#16769) was purchased from Cell Signaling Technology.. Cycloheximide (HY-12320), MG-132 (HY-13259), Chloroquine (HY-17589A), R-7050 (HY-110203), Shancigusin I (HY-N8183), Rebaudioside F (HY-N6887), Jionoside B1 (HY-N2218), and Carnostatine hydrochloride (HY-112431A) were obtained from MedChemExpress.

    Inhibition:

    Article Title: Low-dose TNF-α drives malignant progression and lipid metabolism in glioblastoma through the TRAF2-FASN axis
    Article Snippet: The recombinant human TNF-α protein (#16769) was purchased from Cell Signaling Technology.The recombinant human TNF-α protein (#16769) was purchased from Cell Signaling Technology.. Cycloheximide (HY-12320), MG-132 (HY-13259), Chloroquine (HY-17589A), R-7050 (HY-110203), Shancigusin I (HY-N8183), Rebaudioside F (HY-N6887), Jionoside B1 (HY-N2218), and Carnostatine hydrochloride (HY-112431A) were obtained from MedChemExpress.

    Luciferase:

    Article Title: Low-dose TNF-α drives malignant progression and lipid metabolism in glioblastoma through the TRAF2-FASN axis
    Article Snippet: The recombinant human TNF-α protein (#16769) was purchased from Cell Signaling Technology.The recombinant human TNF-α protein (#16769) was purchased from Cell Signaling Technology.. Cycloheximide (HY-12320), MG-132 (HY-13259), Chloroquine (HY-17589A), R-7050 (HY-110203), Shancigusin I (HY-N8183), Rebaudioside F (HY-N6887), Jionoside B1 (HY-N2218), and Carnostatine hydrochloride (HY-112431A) were obtained from MedChemExpress.

    In Vivo Imaging:

    Article Title: Low-dose TNF-α drives malignant progression and lipid metabolism in glioblastoma through the TRAF2-FASN axis
    Article Snippet: The recombinant human TNF-α protein (#16769) was purchased from Cell Signaling Technology.The recombinant human TNF-α protein (#16769) was purchased from Cell Signaling Technology.. Cycloheximide (HY-12320), MG-132 (HY-13259), Chloroquine (HY-17589A), R-7050 (HY-110203), Shancigusin I (HY-N8183), Rebaudioside F (HY-N6887), Jionoside B1 (HY-N2218), and Carnostatine hydrochloride (HY-112431A) were obtained from MedChemExpress.



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    A Flowchart of mass spectrometry analysis to identify proteins interacting with TRAF2 in U251 cells. B The interaction between endogenous TRAF2 and FASN was verified in U251 and A172 cells by co-immunoprecipitation (Co-IP) assay. C Co-immunoprecipitation (Co-IP) assay was performed to detect the interaction between TRAF2 and FASN in HEK293T cells co-transfected with His-TRAF2 and Flag-FASN. D IF staining analysis confirmed the cytoplasmic co-localization of TRAF2 and FASN proteins in U251 and A172 cells. Endogenous TRAF2 and FASN were stained with anti-TRAF2 (green) and anti-FASN (red) antibodies, respectively. Scale bar, 20 μm. E Western blot analysis of FASN protein expression in U251 and A172 cells overexpressing TRAF2. F Western blot analysis of FASN protein expression in U251 and A172 cells with TRAF2 knockdown. G FASN protein expression was assessed by Western blot in HEK293T cells transfected with Flag-FASN and increasing doses of His-TRAF2 plasmid. H U251 and A172 cells were transiently transfected to express His-TRAF2. Immunofluorescence analysis was performed using anti-His-TRAF2 (green) and anti-FASN (red) antibodies. Nuclei were stained with Hoechst (blue). White arrows indicate His-TRAF2-positive cells. Scale bar, 30 μm. I Western blot analysis of lysates from U251 and A172 cells with TRAF2 knockdown treated with DMSO, MG132 (10 μM), or chloroquine (25 μM). J RT-qPCR analysis of FASN mRNA expression levels in U251 and A172 cells with TRAF2 overexpression or knockdown ( n = 5, Student’s t -test, ns not significant). K <t>Cycloheximide</t> (CHX, 100 μg/ml) was used to treat U251 and A172 cells overexpressing TRAF2 to block new protein synthesis and measure the protein half-life of FASN. Protein stability was analyzed using ImageJ ( n = 5, Student’s t -test, ** p < 0.01). L Cycloheximide (CHX, 100 μg/ml) was used to treat U251 and A172 cells with TRAF2 knockdown to block new protein synthesis and measure the protein half-life of FASN. Protein stability was analyzed using ImageJ ( n = 5, Student’s t -test, ** p < 0.01).
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    A Flowchart of mass spectrometry analysis to identify proteins interacting with TRAF2 in U251 cells. B The interaction between endogenous TRAF2 and FASN was verified in U251 and A172 cells by co-immunoprecipitation (Co-IP) assay. C Co-immunoprecipitation (Co-IP) assay was performed to detect the interaction between TRAF2 and FASN in HEK293T cells co-transfected with His-TRAF2 and Flag-FASN. D IF staining analysis confirmed the cytoplasmic co-localization of TRAF2 and FASN proteins in U251 and A172 cells. Endogenous TRAF2 and FASN were stained with anti-TRAF2 (green) and anti-FASN (red) antibodies, respectively. Scale bar, 20 μm. E Western blot analysis of FASN protein expression in U251 and A172 cells overexpressing TRAF2. F Western blot analysis of FASN protein expression in U251 and A172 cells with TRAF2 knockdown. G FASN protein expression was assessed by Western blot in HEK293T cells transfected with Flag-FASN and increasing doses of His-TRAF2 plasmid. H U251 and A172 cells were transiently transfected to express His-TRAF2. Immunofluorescence analysis was performed using anti-His-TRAF2 (green) and anti-FASN (red) antibodies. Nuclei were stained with Hoechst (blue). White arrows indicate His-TRAF2-positive cells. Scale bar, 30 μm. I Western blot analysis of lysates from U251 and A172 cells with TRAF2 knockdown treated with DMSO, MG132 (10 μM), or chloroquine (25 μM). J RT-qPCR analysis of FASN mRNA expression levels in U251 and A172 cells with TRAF2 overexpression or knockdown ( n = 5, Student’s t -test, ns not significant). K Cycloheximide (CHX, 100 μg/ml) was used to treat U251 and A172 cells overexpressing TRAF2 to block new protein synthesis and measure the protein half-life of FASN. Protein stability was analyzed using ImageJ ( n = 5, Student’s t -test, ** p < 0.01). L Cycloheximide (CHX, 100 μg/ml) was used to treat U251 and A172 cells with TRAF2 knockdown to block new protein synthesis and measure the protein half-life of FASN. Protein stability was analyzed using ImageJ ( n = 5, Student’s t -test, ** p < 0.01).

    Journal: Cell Death Discovery

    Article Title: Low-dose TNF-α drives malignant progression and lipid metabolism in glioblastoma through the TRAF2-FASN axis

    doi: 10.1038/s41420-026-03087-x

    Figure Lengend Snippet: A Flowchart of mass spectrometry analysis to identify proteins interacting with TRAF2 in U251 cells. B The interaction between endogenous TRAF2 and FASN was verified in U251 and A172 cells by co-immunoprecipitation (Co-IP) assay. C Co-immunoprecipitation (Co-IP) assay was performed to detect the interaction between TRAF2 and FASN in HEK293T cells co-transfected with His-TRAF2 and Flag-FASN. D IF staining analysis confirmed the cytoplasmic co-localization of TRAF2 and FASN proteins in U251 and A172 cells. Endogenous TRAF2 and FASN were stained with anti-TRAF2 (green) and anti-FASN (red) antibodies, respectively. Scale bar, 20 μm. E Western blot analysis of FASN protein expression in U251 and A172 cells overexpressing TRAF2. F Western blot analysis of FASN protein expression in U251 and A172 cells with TRAF2 knockdown. G FASN protein expression was assessed by Western blot in HEK293T cells transfected with Flag-FASN and increasing doses of His-TRAF2 plasmid. H U251 and A172 cells were transiently transfected to express His-TRAF2. Immunofluorescence analysis was performed using anti-His-TRAF2 (green) and anti-FASN (red) antibodies. Nuclei were stained with Hoechst (blue). White arrows indicate His-TRAF2-positive cells. Scale bar, 30 μm. I Western blot analysis of lysates from U251 and A172 cells with TRAF2 knockdown treated with DMSO, MG132 (10 μM), or chloroquine (25 μM). J RT-qPCR analysis of FASN mRNA expression levels in U251 and A172 cells with TRAF2 overexpression or knockdown ( n = 5, Student’s t -test, ns not significant). K Cycloheximide (CHX, 100 μg/ml) was used to treat U251 and A172 cells overexpressing TRAF2 to block new protein synthesis and measure the protein half-life of FASN. Protein stability was analyzed using ImageJ ( n = 5, Student’s t -test, ** p < 0.01). L Cycloheximide (CHX, 100 μg/ml) was used to treat U251 and A172 cells with TRAF2 knockdown to block new protein synthesis and measure the protein half-life of FASN. Protein stability was analyzed using ImageJ ( n = 5, Student’s t -test, ** p < 0.01).

    Article Snippet: Cycloheximide (HY-12320), MG-132 (HY-13259), Chloroquine (HY-17589A), R-7050 (HY-110203), Shancigusin I (HY-N8183), Rebaudioside F (HY-N6887), Jionoside B1 (HY-N2218), and Carnostatine hydrochloride (HY-112431A) were obtained from MedChemExpress.

    Techniques: Mass Spectrometry, Co-Immunoprecipitation Assay, Transfection, Staining, Western Blot, Expressing, Knockdown, Plasmid Preparation, Immunofluorescence, Quantitative RT-PCR, Over Expression, Blocking Assay

    A U251 and A172 cells were treated with TNF-α (10 ng/mL) for indicated durations, and endogenous FASN and TRAF2 protein levels were analyzed by Western blot. B U251 and A172 cells stimulated with TNF-α (10 ng/mL) were treated with cycloheximide (CHX, 100 μg/mL) to block new protein synthesis. The protein half-life of FASN was measured and protein stability was analyzed using ImageJ ( n = 5, Student’s t -test, ** p < 0.01). C U251 and A172 cells stimulated with TNF-α (10 ng/mL) were treated with cycloheximide (CHX, 100 μg/mL) to block new protein synthesis. The protein half-life of TRAF2 was measured and protein stability was analyzed using ImageJ ( n = 5, Student’s t -test, ns: not significant). D RT-qPCR analysis of FASN mRNA expression levels in U251 and A172 cells treated with TNF-α (10 ng/mL) for 24 h ( n = 5, Student’s t -test, ns: not significant). E Endogenous FASN and TRAF2 protein levels were detected by Western blot in TRAF2-knockdown U251 and A172 cells re-exposed to TNF-α (10 ng/mL) stimulation. F Co-immunoprecipitation analysis of TRAF2 and FASN in U251 and A172 cells after stimulation with TNF-α (10 ng/mL) for indicated durations. G HEK293T cells co-transfected with HIS-TRAF2 and FLAG-FASN plasmids were treated with TNF-α (10 ng/mL) for indicated durations. Co-immunoprecipitation (Co-IP) was performed to assess the effect of TNF-α on the TRAF2-FASN interaction. H , I Levels of total ubiquitination and K63-linked polyubiquitination co-immunoprecipitated with TRAF2 were detected in U251 and A172 cells stimulated with TNF-α (10 ng/mL). J HEK293T cells co-transfected with HIS-TRAF2 and HA-Ub (WT or K63-only) plasmids were treated with or without TNF-α (10 ng/mL) stimulation. Immunoprecipitation was performed using an anti-HIS antibody to detect TRAF2 ubiquitination levels. K , L Levels of total ubiquitination and K63-linked polyubiquitination co-immunoprecipitated with FASN were detected in U251 and A172 cells stimulated with TNF-α (10 ng/mL). M HEK293T cells co-transfected with FLAG-FASN, HA-Ub (WT or K63-only), and HIS-TRAF2 plasmids were treated with or without TNF-α (10 ng/mL) stimulation. Immunoprecipitation was performed using an anti-FLAG antibody to detect FASN ubiquitination levels. N TRAF2 knockdown abolished the TNF-α-induced upregulation of endogenous FASN ubiquitination and protein levels in U251 and A172 cells.

    Journal: Cell Death Discovery

    Article Title: Low-dose TNF-α drives malignant progression and lipid metabolism in glioblastoma through the TRAF2-FASN axis

    doi: 10.1038/s41420-026-03087-x

    Figure Lengend Snippet: A U251 and A172 cells were treated with TNF-α (10 ng/mL) for indicated durations, and endogenous FASN and TRAF2 protein levels were analyzed by Western blot. B U251 and A172 cells stimulated with TNF-α (10 ng/mL) were treated with cycloheximide (CHX, 100 μg/mL) to block new protein synthesis. The protein half-life of FASN was measured and protein stability was analyzed using ImageJ ( n = 5, Student’s t -test, ** p < 0.01). C U251 and A172 cells stimulated with TNF-α (10 ng/mL) were treated with cycloheximide (CHX, 100 μg/mL) to block new protein synthesis. The protein half-life of TRAF2 was measured and protein stability was analyzed using ImageJ ( n = 5, Student’s t -test, ns: not significant). D RT-qPCR analysis of FASN mRNA expression levels in U251 and A172 cells treated with TNF-α (10 ng/mL) for 24 h ( n = 5, Student’s t -test, ns: not significant). E Endogenous FASN and TRAF2 protein levels were detected by Western blot in TRAF2-knockdown U251 and A172 cells re-exposed to TNF-α (10 ng/mL) stimulation. F Co-immunoprecipitation analysis of TRAF2 and FASN in U251 and A172 cells after stimulation with TNF-α (10 ng/mL) for indicated durations. G HEK293T cells co-transfected with HIS-TRAF2 and FLAG-FASN plasmids were treated with TNF-α (10 ng/mL) for indicated durations. Co-immunoprecipitation (Co-IP) was performed to assess the effect of TNF-α on the TRAF2-FASN interaction. H , I Levels of total ubiquitination and K63-linked polyubiquitination co-immunoprecipitated with TRAF2 were detected in U251 and A172 cells stimulated with TNF-α (10 ng/mL). J HEK293T cells co-transfected with HIS-TRAF2 and HA-Ub (WT or K63-only) plasmids were treated with or without TNF-α (10 ng/mL) stimulation. Immunoprecipitation was performed using an anti-HIS antibody to detect TRAF2 ubiquitination levels. K , L Levels of total ubiquitination and K63-linked polyubiquitination co-immunoprecipitated with FASN were detected in U251 and A172 cells stimulated with TNF-α (10 ng/mL). M HEK293T cells co-transfected with FLAG-FASN, HA-Ub (WT or K63-only), and HIS-TRAF2 plasmids were treated with or without TNF-α (10 ng/mL) stimulation. Immunoprecipitation was performed using an anti-FLAG antibody to detect FASN ubiquitination levels. N TRAF2 knockdown abolished the TNF-α-induced upregulation of endogenous FASN ubiquitination and protein levels in U251 and A172 cells.

    Article Snippet: Cycloheximide (HY-12320), MG-132 (HY-13259), Chloroquine (HY-17589A), R-7050 (HY-110203), Shancigusin I (HY-N8183), Rebaudioside F (HY-N6887), Jionoside B1 (HY-N2218), and Carnostatine hydrochloride (HY-112431A) were obtained from MedChemExpress.

    Techniques: Western Blot, Blocking Assay, Quantitative RT-PCR, Expressing, Knockdown, Immunoprecipitation, Transfection, Co-Immunoprecipitation Assay, Ubiquitin Proteomics

    A Simulated binding conformations of candidate small-molecule compounds obtained through virtual screening targeting the RING domain essential for TRAF2 catalytic activity. B Endogenous FASN and TRAF2 protein levels were analyzed by Western blot in U251 and A172 cells treated with gradient concentrations of Jionoside B1 for 24 h. C Endogenous FASN and TRAF2 protein levels were detected by Western blot in U251 and A172 cells treated with Jionoside B1 (100 μM) for indicated durations. D U251 and A172 cells stimulated with Jionoside B1 (100 μM) were treated with cycloheximide (CHX, 100 μg/mL) to block new protein synthesis. The protein half-life of FASN was measured and protein stability was analyzed using ImageJ ( n = 5, Student’s t -test, ** p < 0.01). E U251 and A172 cells stimulated with Jionoside B1 (100 μM) were treated with cycloheximide (CHX, 100 μg/mL) to block new protein synthesis. The protein half-life of TRAF2 was measured and protein stability was analyzed using ImageJ ( n = 5, Student’s t -test, ns: not significant). F . Endogenous FASN protein levels were detected by Western blot in TRAF2-overexpressing U251 and A172 cells re-exposed to Jionoside B1 (100 μM) stimulation. G U251 and A172 cells were treated with TNF-α (10 ng/mL) alone or in combination with Jionoside B1 (100 μM) for 24 hours. H , I Levels of total ubiquitination and K63-linked polyubiquitination co-immunoprecipitated with FASN were detected in U251 and A172 cells stimulated with Jionoside B1 (100 μM). J . HEK293T cells co-transfected with FLAG-FASN and HA-Ub (K63-only) plasmids were treated with or without Jionoside B1 (100 μM) stimulation. Immunoprecipitation was performed using an anti-FLAG antibody to detect K63-linked ubiquitination of FASN. K Co-immunoprecipitation analysis of TRAF2 and FASN in U251 and A172 cells after stimulation with Jionoside B1 (100 μM) for indicated durations. L HEK293T cells co-transfected with HIS-TRAF2 and FLAG-FASN plasmids were treated with Jionoside B1 (100 μM) for indicated durations. Co-immunoprecipitation (Co-IP) was performed to assess the effect of Jionoside B1 on the TRAF2-FASN interaction. M Schematic diagram of in vivo evaluation of Jionoside B1’s tumor growth inhibition. Luciferase-expressing U251 cells were implanted into the brains of nude mice, and intraperitoneal injections of DMSO or Jionoside B1 (20 mg/kg/day) were administered every 2 days starting from day 14 post-surgery. Tumor volume was monitored by in vivo imaging on days 14 and 28 post-surgery. N In vivo imaging of orthotopic intracranial tumors. O Statistical results of tumor volumes ( n = 4, Student’s t -test, *** p < 0.001).

    Journal: Cell Death Discovery

    Article Title: Low-dose TNF-α drives malignant progression and lipid metabolism in glioblastoma through the TRAF2-FASN axis

    doi: 10.1038/s41420-026-03087-x

    Figure Lengend Snippet: A Simulated binding conformations of candidate small-molecule compounds obtained through virtual screening targeting the RING domain essential for TRAF2 catalytic activity. B Endogenous FASN and TRAF2 protein levels were analyzed by Western blot in U251 and A172 cells treated with gradient concentrations of Jionoside B1 for 24 h. C Endogenous FASN and TRAF2 protein levels were detected by Western blot in U251 and A172 cells treated with Jionoside B1 (100 μM) for indicated durations. D U251 and A172 cells stimulated with Jionoside B1 (100 μM) were treated with cycloheximide (CHX, 100 μg/mL) to block new protein synthesis. The protein half-life of FASN was measured and protein stability was analyzed using ImageJ ( n = 5, Student’s t -test, ** p < 0.01). E U251 and A172 cells stimulated with Jionoside B1 (100 μM) were treated with cycloheximide (CHX, 100 μg/mL) to block new protein synthesis. The protein half-life of TRAF2 was measured and protein stability was analyzed using ImageJ ( n = 5, Student’s t -test, ns: not significant). F . Endogenous FASN protein levels were detected by Western blot in TRAF2-overexpressing U251 and A172 cells re-exposed to Jionoside B1 (100 μM) stimulation. G U251 and A172 cells were treated with TNF-α (10 ng/mL) alone or in combination with Jionoside B1 (100 μM) for 24 hours. H , I Levels of total ubiquitination and K63-linked polyubiquitination co-immunoprecipitated with FASN were detected in U251 and A172 cells stimulated with Jionoside B1 (100 μM). J . HEK293T cells co-transfected with FLAG-FASN and HA-Ub (K63-only) plasmids were treated with or without Jionoside B1 (100 μM) stimulation. Immunoprecipitation was performed using an anti-FLAG antibody to detect K63-linked ubiquitination of FASN. K Co-immunoprecipitation analysis of TRAF2 and FASN in U251 and A172 cells after stimulation with Jionoside B1 (100 μM) for indicated durations. L HEK293T cells co-transfected with HIS-TRAF2 and FLAG-FASN plasmids were treated with Jionoside B1 (100 μM) for indicated durations. Co-immunoprecipitation (Co-IP) was performed to assess the effect of Jionoside B1 on the TRAF2-FASN interaction. M Schematic diagram of in vivo evaluation of Jionoside B1’s tumor growth inhibition. Luciferase-expressing U251 cells were implanted into the brains of nude mice, and intraperitoneal injections of DMSO or Jionoside B1 (20 mg/kg/day) were administered every 2 days starting from day 14 post-surgery. Tumor volume was monitored by in vivo imaging on days 14 and 28 post-surgery. N In vivo imaging of orthotopic intracranial tumors. O Statistical results of tumor volumes ( n = 4, Student’s t -test, *** p < 0.001).

    Article Snippet: Cycloheximide (HY-12320), MG-132 (HY-13259), Chloroquine (HY-17589A), R-7050 (HY-110203), Shancigusin I (HY-N8183), Rebaudioside F (HY-N6887), Jionoside B1 (HY-N2218), and Carnostatine hydrochloride (HY-112431A) were obtained from MedChemExpress.

    Techniques: Binding Assay, Activity Assay, Western Blot, Blocking Assay, Ubiquitin Proteomics, Immunoprecipitation, Transfection, Co-Immunoprecipitation Assay, In Vivo, Inhibition, Luciferase, Expressing, In Vivo Imaging