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anti impdh2  (Proteintech)


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    Structured Review

    Proteintech anti impdh2
    Anti Impdh2, supplied by Proteintech, used in various techniques. Bioz Stars score: 95/100, based on 71 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/anti+impdh2+antibody/IMPDH2+Antibody/pm41922317-48-10-11
    Average 95 stars, based on 71 article reviews
    anti impdh2 - by Bioz Stars, 2026-10
    95/100 stars

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    Related Articles

    Incubation:

    Article Title: Circular RNA circPFKP promotes cell proliferation by activating IMPDH2 in prostate cancer.
    Article Snippet: Prostate cancer (PCa), especially castration-resistant PCa, is a common and fatal disease. circRNAs had been confirmed to affect the proliferation of a variety of malignant tumors.. Exploring the role of circRNAs in PCa progression and discovering new therapeutic targets are of great importance for the treatment of PCa.. In the present study, we found that the expression of circPFKP was significantly increased in PCa tissues compared with adjacent noncancerous prostate tissues, and was correlated with the D’Amico risk classification, N stage, and prognostic stage group of PCa.

    Article Title: Berberrubine is a novel and selective IMPDH2 inhibitor that impairs the growth of colorectal cancer.
    Article Snippet: Inosine monophosphate dehydrogenase (IMPDH) catalyzes the rate-limiting reaction in the de novo synthesis pathway of guanine nucleotides that is highly required for cancer cell outgrowth.. Herein, we found that IMPDH isoform 2 (IMPDH2) is highly expressed in colorectal cancer (CRC) and is correlated with poor patient prognosis.. Via structure-based virtual screening, we identified berberrubine, a critical ingredient of the medical plant Coptis chinensis, as a novel, selective, and competitive inhibitor of IMPDH2, which demonstrated over 15-fold selectivity to IMPDH2 than IMPDH1.

    Staining:

    Article Title: Anti-rods/rings autoantibodies in a patient with pancreatic injury.
    Article Snippet: Identifying the clinical implication of autoantibodies is an important aspect of studies on autoimmunity.. Autoantibodies against rods/rings (anti-RR) are rare, but are found mainly in patients with hepatitis C who have been treated with interferon-α plus ribavirin combination therapy (IFN/RBV) [1–3].. However, it is not clear why or how these autoantibodies are formed, or whether they are clinically significant.

    Western Blot:

    Article Title: FDA-approved IMPDH inhibitors synergize with ribavirin to inhibit respiratory syncytial virus by interfering with purine de novo synthesis.
    Article Snippet: Immunoreactive bands were detected using a Hypersensitive ECL Chemiluminescence Kit (NCM, P10300) and imaged on a Tanon 5200 imaging system. .. The following primary antibodies were used for Western blot analysis: anti-RSV F antibody (Sino Biological, 11049-R302, 1:2000), anti-IMPDH2 antibody (Proteintech, 67663, 1:2000) and anti-HRP GAPDH (Proteintech, HRP-60004,1:5000). ..

    Article Title: FDA-approved IMPDH inhibitors synergize with ribavirin to inhibit respiratory syncytial virus by interfering with purine de novo synthesis
    Article Snippet: Immunoreactive bands were detected using a Hypersensitive ECL Chemiluminescence Kit (NCM, P10300 ) and imaged on a Tanon 5200 imaging system. .. The following primary antibodies were used for Western blot analysis: anti-RSV F antibody (Sino Biological, 11049-R302, 1:2000), anti-IMPDH2 antibody (Proteintech, 67663, 1:2000) and anti-HRP GAPDH (Proteintech, HRP-60004, 1:5000). ..

    Analysis:

    Article Title: FDA-approved IMPDH inhibitors synergize with ribavirin to inhibit respiratory syncytial virus by interfering with purine de novo synthesis.
    Article Snippet: Immunoreactive bands were detected using a Hypersensitive ECL Chemiluminescence Kit (NCM, P10300) and imaged on a Tanon 5200 imaging system. .. The following primary antibodies were used for Western blot analysis: anti-RSV F antibody (Sino Biological, 11049-R302, 1:2000), anti-IMPDH2 antibody (Proteintech, 67663, 1:2000) and anti-HRP GAPDH (Proteintech, HRP-60004,1:5000). ..

    Article Title: FDA-approved IMPDH inhibitors synergize with ribavirin to inhibit respiratory syncytial virus by interfering with purine de novo synthesis
    Article Snippet: Immunoreactive bands were detected using a Hypersensitive ECL Chemiluminescence Kit (NCM, P10300 ) and imaged on a Tanon 5200 imaging system. .. The following primary antibodies were used for Western blot analysis: anti-RSV F antibody (Sino Biological, 11049-R302, 1:2000), anti-IMPDH2 antibody (Proteintech, 67663, 1:2000) and anti-HRP GAPDH (Proteintech, HRP-60004, 1:5000). ..

    Immunolabeling:

    Article Title: A simple Fourier filter for suppression of the missing wedge ray artefacts in single-axis electron tomographic reconstructions
    Article Snippet: .. The cells were fixed in formaldehyde, permeabilized and immunolabeled with the primary anti-IMPDH2 antibody (12948-1-AP, Proteintech, Manchester, United Kingdom) and the secondary antibody conjugated with ultrasmall gold (Aurion, Wageningen, The Netherlands). .. After the pre-embedding, the ultrasmall colloidal gold particles were silver enhanced, the immunolabeled cells were postfixed with glutaraldehyde, incubated with R-GENT SE-EM (Aurion), dehydrated in ethanol, embedded into Araldite/Embed 812 and polymerized.



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    ( A – D ) Representative immunohistochemical images showing expression of ADSS1 ( A ) and <t>IMPDH2</t> ( B ) in the induced membrane. Quantification of ADSS1 ( C ) and IMPDH2 ( D ) staining using ImageJ. Each dot represents one animal (IM, n = 6; BD, n = 6). Data are presented as area fraction and normalized to DAPI areas. Box plots represent the median (line), interquartile range (box), and minimum to maximum values (whiskers). P < 0.05 was considered statistically significant. ( E ) Summary of showing alterations in serum and induced membrane at week 6 in IMT rats. Scale bars: 50 μm. ADSS1, adenylosuccinate synthase 1; IMPDH2, inosine monophosphate dehydrogenase 2; BD, bone defect; IM, induced membrane.
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    anti impdh2 mab  (ABclonal Biotechnology)
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    (A–B) Impact of JEV infection on protein expression of key DNPB enzymes. N2a cells were infected or mock-infected with JEV at an MOI of 1. The expression of PFAS, GART, IMPDH1, <t>IMPDH2,</t> ADSS, GMPS, and β-Tubulin proteins were detected by immunoblotting, and representative blots from 3 independent experiments are presented (A) . Protein levels were quantified by using Image J software, normalized to β-Tubulin, and presented relative to the mock-infected control (n = 3) (B) . (C–E) Inhibitory effect of MPA on JEV replication. N2a cells were infected with JEV at an MOI of 5, followed by treatment with either 50 nM MPA or an equivalent volume of DMSO at 1.5 hpi. The levels of intracellular viral RNA (C) and E protein (D) , as well as the infectious virus production in the supernatant (E) at 6, 12, 18, and 24 hpi, were determined using qRT-PCR, immunoblotting, and plaque assay, respectively. (F–H) The effect of guanosine (Gua) treatment on MPA-mediated inhibition of JEV replication. N2a cells infected with JEV were treated with either MPA or DMSO, with or without addition of 100 μM Gua. The levels of intracellular viral RNA (F) and E protein (G) , as well as the production of infectious virus in the supernatant (H) , were determined using qRT-PCR, immunoblotting, and plaque assay, respectively, at 18 hpi. (I–J) Inhibitory effect of Ag2037 on JEV replication. N2a cells were infected with JEV at an MOI of 5, followed by treatment with either 0.5 μM Ag2037 or an equivalent volume of DMSO at 1.5 hpi. The levels of intracellular viral RNA (I) and viral titers in the supernatant (J) were determined at 6, 12, 18, and 24 hpi. (K–L) The effect of hypoxanthine (Hyp) treatment on Ag2037-mediated inhibition of JEV replication. N2a cells infected with JEV were treated with either Ag2037 or DMSO, with or without addition of Hyp at 1.5 hpi. The levels of intracellular viral RNA (K) and viral titers in the supernatant (L) were measured at 18 hpi. Data are presented as mean ± s.e.m. from 3 independent biological replicates (n = 3). Significance was determined using two-tailed unpaired Student’s t-test (B) , one-way (F, H, K and L) or two-way ANOVA (C, E, I and J) (*p < 0.05, **p < 0.01, ***p < 0.001; ****p < 0.0001; ns, not significant).
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    (A–B) Impact of JEV infection on protein expression of key DNPB enzymes. N2a cells were infected or mock-infected with JEV at an MOI of 1. The expression of PFAS, GART, IMPDH1, <t>IMPDH2,</t> ADSS, GMPS, and β-Tubulin proteins were detected by immunoblotting, and representative blots from 3 independent experiments are presented (A) . Protein levels were quantified by using Image J software, normalized to β-Tubulin, and presented relative to the mock-infected control (n = 3) (B) . (C–E) Inhibitory effect of MPA on JEV replication. N2a cells were infected with JEV at an MOI of 5, followed by treatment with either 50 nM MPA or an equivalent volume of DMSO at 1.5 hpi. The levels of intracellular viral RNA (C) and E protein (D) , as well as the infectious virus production in the supernatant (E) at 6, 12, 18, and 24 hpi, were determined using qRT-PCR, immunoblotting, and plaque assay, respectively. (F–H) The effect of guanosine (Gua) treatment on MPA-mediated inhibition of JEV replication. N2a cells infected with JEV were treated with either MPA or DMSO, with or without addition of 100 μM Gua. The levels of intracellular viral RNA (F) and E protein (G) , as well as the production of infectious virus in the supernatant (H) , were determined using qRT-PCR, immunoblotting, and plaque assay, respectively, at 18 hpi. (I–J) Inhibitory effect of Ag2037 on JEV replication. N2a cells were infected with JEV at an MOI of 5, followed by treatment with either 0.5 μM Ag2037 or an equivalent volume of DMSO at 1.5 hpi. The levels of intracellular viral RNA (I) and viral titers in the supernatant (J) were determined at 6, 12, 18, and 24 hpi. (K–L) The effect of hypoxanthine (Hyp) treatment on Ag2037-mediated inhibition of JEV replication. N2a cells infected with JEV were treated with either Ag2037 or DMSO, with or without addition of Hyp at 1.5 hpi. The levels of intracellular viral RNA (K) and viral titers in the supernatant (L) were measured at 18 hpi. Data are presented as mean ± s.e.m. from 3 independent biological replicates (n = 3). Significance was determined using two-tailed unpaired Student’s t-test (B) , one-way (F, H, K and L) or two-way ANOVA (C, E, I and J) (*p < 0.05, **p < 0.01, ***p < 0.001; ****p < 0.0001; ns, not significant).
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    (A–B) Impact of JEV infection on protein expression of key DNPB enzymes. N2a cells were infected or mock-infected with JEV at an MOI of 1. The expression of PFAS, GART, IMPDH1, <t>IMPDH2,</t> ADSS, GMPS, and β-Tubulin proteins were detected by immunoblotting, and representative blots from 3 independent experiments are presented (A) . Protein levels were quantified by using Image J software, normalized to β-Tubulin, and presented relative to the mock-infected control (n = 3) (B) . (C–E) Inhibitory effect of MPA on JEV replication. N2a cells were infected with JEV at an MOI of 5, followed by treatment with either 50 nM MPA or an equivalent volume of DMSO at 1.5 hpi. The levels of intracellular viral RNA (C) and E protein (D) , as well as the infectious virus production in the supernatant (E) at 6, 12, 18, and 24 hpi, were determined using qRT-PCR, immunoblotting, and plaque assay, respectively. (F–H) The effect of guanosine (Gua) treatment on MPA-mediated inhibition of JEV replication. N2a cells infected with JEV were treated with either MPA or DMSO, with or without addition of 100 μM Gua. The levels of intracellular viral RNA (F) and E protein (G) , as well as the production of infectious virus in the supernatant (H) , were determined using qRT-PCR, immunoblotting, and plaque assay, respectively, at 18 hpi. (I–J) Inhibitory effect of Ag2037 on JEV replication. N2a cells were infected with JEV at an MOI of 5, followed by treatment with either 0.5 μM Ag2037 or an equivalent volume of DMSO at 1.5 hpi. The levels of intracellular viral RNA (I) and viral titers in the supernatant (J) were determined at 6, 12, 18, and 24 hpi. (K–L) The effect of hypoxanthine (Hyp) treatment on Ag2037-mediated inhibition of JEV replication. N2a cells infected with JEV were treated with either Ag2037 or DMSO, with or without addition of Hyp at 1.5 hpi. The levels of intracellular viral RNA (K) and viral titers in the supernatant (L) were measured at 18 hpi. Data are presented as mean ± s.e.m. from 3 independent biological replicates (n = 3). Significance was determined using two-tailed unpaired Student’s t-test (B) , one-way (F, H, K and L) or two-way ANOVA (C, E, I and J) (*p < 0.05, **p < 0.01, ***p < 0.001; ****p < 0.0001; ns, not significant).
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    (A–B) Impact of JEV infection on protein expression of key DNPB enzymes. N2a cells were infected or mock-infected with JEV at an MOI of 1. The expression of PFAS, GART, IMPDH1, <t>IMPDH2,</t> ADSS, GMPS, and β-Tubulin proteins were detected by immunoblotting, and representative blots from 3 independent experiments are presented (A) . Protein levels were quantified by using Image J software, normalized to β-Tubulin, and presented relative to the mock-infected control (n = 3) (B) . (C–E) Inhibitory effect of MPA on JEV replication. N2a cells were infected with JEV at an MOI of 5, followed by treatment with either 50 nM MPA or an equivalent volume of DMSO at 1.5 hpi. The levels of intracellular viral RNA (C) and E protein (D) , as well as the infectious virus production in the supernatant (E) at 6, 12, 18, and 24 hpi, were determined using qRT-PCR, immunoblotting, and plaque assay, respectively. (F–H) The effect of guanosine (Gua) treatment on MPA-mediated inhibition of JEV replication. N2a cells infected with JEV were treated with either MPA or DMSO, with or without addition of 100 μM Gua. The levels of intracellular viral RNA (F) and E protein (G) , as well as the production of infectious virus in the supernatant (H) , were determined using qRT-PCR, immunoblotting, and plaque assay, respectively, at 18 hpi. (I–J) Inhibitory effect of Ag2037 on JEV replication. N2a cells were infected with JEV at an MOI of 5, followed by treatment with either 0.5 μM Ag2037 or an equivalent volume of DMSO at 1.5 hpi. The levels of intracellular viral RNA (I) and viral titers in the supernatant (J) were determined at 6, 12, 18, and 24 hpi. (K–L) The effect of hypoxanthine (Hyp) treatment on Ag2037-mediated inhibition of JEV replication. N2a cells infected with JEV were treated with either Ag2037 or DMSO, with or without addition of Hyp at 1.5 hpi. The levels of intracellular viral RNA (K) and viral titers in the supernatant (L) were measured at 18 hpi. Data are presented as mean ± s.e.m. from 3 independent biological replicates (n = 3). Significance was determined using two-tailed unpaired Student’s t-test (B) , one-way (F, H, K and L) or two-way ANOVA (C, E, I and J) (*p < 0.05, **p < 0.01, ***p < 0.001; ****p < 0.0001; ns, not significant).
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    anti impdh2 antibody  (Proteintech)
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    (A–B) Impact of JEV infection on protein expression of key DNPB enzymes. N2a cells were infected or mock-infected with JEV at an MOI of 1. The expression of PFAS, GART, IMPDH1, <t>IMPDH2,</t> ADSS, GMPS, and β-Tubulin proteins were detected by immunoblotting, and representative blots from 3 independent experiments are presented (A) . Protein levels were quantified by using Image J software, normalized to β-Tubulin, and presented relative to the mock-infected control (n = 3) (B) . (C–E) Inhibitory effect of MPA on JEV replication. N2a cells were infected with JEV at an MOI of 5, followed by treatment with either 50 nM MPA or an equivalent volume of DMSO at 1.5 hpi. The levels of intracellular viral RNA (C) and E protein (D) , as well as the infectious virus production in the supernatant (E) at 6, 12, 18, and 24 hpi, were determined using qRT-PCR, immunoblotting, and plaque assay, respectively. (F–H) The effect of guanosine (Gua) treatment on MPA-mediated inhibition of JEV replication. N2a cells infected with JEV were treated with either MPA or DMSO, with or without addition of 100 μM Gua. The levels of intracellular viral RNA (F) and E protein (G) , as well as the production of infectious virus in the supernatant (H) , were determined using qRT-PCR, immunoblotting, and plaque assay, respectively, at 18 hpi. (I–J) Inhibitory effect of Ag2037 on JEV replication. N2a cells were infected with JEV at an MOI of 5, followed by treatment with either 0.5 μM Ag2037 or an equivalent volume of DMSO at 1.5 hpi. The levels of intracellular viral RNA (I) and viral titers in the supernatant (J) were determined at 6, 12, 18, and 24 hpi. (K–L) The effect of hypoxanthine (Hyp) treatment on Ag2037-mediated inhibition of JEV replication. N2a cells infected with JEV were treated with either Ag2037 or DMSO, with or without addition of Hyp at 1.5 hpi. The levels of intracellular viral RNA (K) and viral titers in the supernatant (L) were measured at 18 hpi. Data are presented as mean ± s.e.m. from 3 independent biological replicates (n = 3). Significance was determined using two-tailed unpaired Student’s t-test (B) , one-way (F, H, K and L) or two-way ANOVA (C, E, I and J) (*p < 0.05, **p < 0.01, ***p < 0.001; ****p < 0.0001; ns, not significant).
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    Average 95 stars, based on 1 article reviews
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    impdh2  (Cell Signaling Technology Inc)
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    Cell Signaling Technology Inc impdh2
    PSMD14 interacts with <t>IMPDH2</t> and regulates its protein stability in GBM. (A) Western blot confirming overexpression of exogenous Flag-tagged PSMD14 in U118MG cells. (B) The Co-IP assay of PSMD14 from U118MG cells. (C) Mass spectrometry identification of proteins co-precipitated with PSMD14 in U118MG cells, followed by intersection analysis to identify common candidate interactors. (D) Bioinformatic overlap of the PSMD14 interactome identified by mass spectrometry with known interactions in the BioGRID database. (E) Network analysis of high-confidence interacting proteins with PSMD14. (F) Western blotting analysis to detect changes in IMPDH2 expression after knockdown of PSMD14. (G) The Co-IP assay confirming the interaction between PSMD14 and IMPDH2 in GBM cells. (H) Structural docking model of PSMD14 and IMPDH2 showing the predicted binding interface rendered with PyMOL (PSMD14 in pink, IMPDH2 in purple). (I) Schematic diagram of the PSMD14 and IMPDH2 domain structures and the design of truncation mutants. (J) The Co-IP analysis using PSMD14 and IMPDH2 truncation mutants, identifying the regions required for their interaction. (K) Cycloheximide (CHX) chase assay in control and PSMD14-knockdown GBM cells, evaluating IMPDH2 protein stability over time. (L) Quantification of IMPDH2 protein half-life from CHX chase assays. Data are presented as mean ± SD (n = 3). *P < 0.05 (one-way ANOVA).
    Impdh2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    ( A – D ) Representative immunohistochemical images showing expression of ADSS1 ( A ) and IMPDH2 ( B ) in the induced membrane. Quantification of ADSS1 ( C ) and IMPDH2 ( D ) staining using ImageJ. Each dot represents one animal (IM, n = 6; BD, n = 6). Data are presented as area fraction and normalized to DAPI areas. Box plots represent the median (line), interquartile range (box), and minimum to maximum values (whiskers). P < 0.05 was considered statistically significant. ( E ) Summary of showing alterations in serum and induced membrane at week 6 in IMT rats. Scale bars: 50 μm. ADSS1, adenylosuccinate synthase 1; IMPDH2, inosine monophosphate dehydrogenase 2; BD, bone defect; IM, induced membrane.

    Journal: JCI Insight

    Article Title: Purine metabolism enhances neovascularization of type H vessels in the induced membrane technique

    doi: 10.1172/jci.insight.200820

    Figure Lengend Snippet: ( A – D ) Representative immunohistochemical images showing expression of ADSS1 ( A ) and IMPDH2 ( B ) in the induced membrane. Quantification of ADSS1 ( C ) and IMPDH2 ( D ) staining using ImageJ. Each dot represents one animal (IM, n = 6; BD, n = 6). Data are presented as area fraction and normalized to DAPI areas. Box plots represent the median (line), interquartile range (box), and minimum to maximum values (whiskers). P < 0.05 was considered statistically significant. ( E ) Summary of showing alterations in serum and induced membrane at week 6 in IMT rats. Scale bars: 50 μm. ADSS1, adenylosuccinate synthase 1; IMPDH2, inosine monophosphate dehydrogenase 2; BD, bone defect; IM, induced membrane.

    Article Snippet: The primary antibodies used included adenylosuccinate synthase 1 (ADSS1; A6516, rabbit polyclonal antibody, ABclonal), inosine monophosphate dehydrogenase 2 (IMPDH2; A15626, rabbit polyclonal antibody, ABclonal), CD31 conjugated with Alexa Flour 488 (#42777, clone 89C2, Cell Signaling), and endomucin (EMCN) conjugated with Alexa Flour 555 (BS-4808R-A555, rabbit polyclonal antibody, Bioss).

    Techniques: Immunohistochemical staining, Expressing, Membrane, Staining

    (A–B) Impact of JEV infection on protein expression of key DNPB enzymes. N2a cells were infected or mock-infected with JEV at an MOI of 1. The expression of PFAS, GART, IMPDH1, IMPDH2, ADSS, GMPS, and β-Tubulin proteins were detected by immunoblotting, and representative blots from 3 independent experiments are presented (A) . Protein levels were quantified by using Image J software, normalized to β-Tubulin, and presented relative to the mock-infected control (n = 3) (B) . (C–E) Inhibitory effect of MPA on JEV replication. N2a cells were infected with JEV at an MOI of 5, followed by treatment with either 50 nM MPA or an equivalent volume of DMSO at 1.5 hpi. The levels of intracellular viral RNA (C) and E protein (D) , as well as the infectious virus production in the supernatant (E) at 6, 12, 18, and 24 hpi, were determined using qRT-PCR, immunoblotting, and plaque assay, respectively. (F–H) The effect of guanosine (Gua) treatment on MPA-mediated inhibition of JEV replication. N2a cells infected with JEV were treated with either MPA or DMSO, with or without addition of 100 μM Gua. The levels of intracellular viral RNA (F) and E protein (G) , as well as the production of infectious virus in the supernatant (H) , were determined using qRT-PCR, immunoblotting, and plaque assay, respectively, at 18 hpi. (I–J) Inhibitory effect of Ag2037 on JEV replication. N2a cells were infected with JEV at an MOI of 5, followed by treatment with either 0.5 μM Ag2037 or an equivalent volume of DMSO at 1.5 hpi. The levels of intracellular viral RNA (I) and viral titers in the supernatant (J) were determined at 6, 12, 18, and 24 hpi. (K–L) The effect of hypoxanthine (Hyp) treatment on Ag2037-mediated inhibition of JEV replication. N2a cells infected with JEV were treated with either Ag2037 or DMSO, with or without addition of Hyp at 1.5 hpi. The levels of intracellular viral RNA (K) and viral titers in the supernatant (L) were measured at 18 hpi. Data are presented as mean ± s.e.m. from 3 independent biological replicates (n = 3). Significance was determined using two-tailed unpaired Student’s t-test (B) , one-way (F, H, K and L) or two-way ANOVA (C, E, I and J) (*p < 0.05, **p < 0.01, ***p < 0.001; ****p < 0.0001; ns, not significant).

    Journal: PLOS Pathogens

    Article Title: Japanese encephalitis virus hijacks the host purine biosynthetic network to promote viral replication in neurons

    doi: 10.1371/journal.ppat.1014335

    Figure Lengend Snippet: (A–B) Impact of JEV infection on protein expression of key DNPB enzymes. N2a cells were infected or mock-infected with JEV at an MOI of 1. The expression of PFAS, GART, IMPDH1, IMPDH2, ADSS, GMPS, and β-Tubulin proteins were detected by immunoblotting, and representative blots from 3 independent experiments are presented (A) . Protein levels were quantified by using Image J software, normalized to β-Tubulin, and presented relative to the mock-infected control (n = 3) (B) . (C–E) Inhibitory effect of MPA on JEV replication. N2a cells were infected with JEV at an MOI of 5, followed by treatment with either 50 nM MPA or an equivalent volume of DMSO at 1.5 hpi. The levels of intracellular viral RNA (C) and E protein (D) , as well as the infectious virus production in the supernatant (E) at 6, 12, 18, and 24 hpi, were determined using qRT-PCR, immunoblotting, and plaque assay, respectively. (F–H) The effect of guanosine (Gua) treatment on MPA-mediated inhibition of JEV replication. N2a cells infected with JEV were treated with either MPA or DMSO, with or without addition of 100 μM Gua. The levels of intracellular viral RNA (F) and E protein (G) , as well as the production of infectious virus in the supernatant (H) , were determined using qRT-PCR, immunoblotting, and plaque assay, respectively, at 18 hpi. (I–J) Inhibitory effect of Ag2037 on JEV replication. N2a cells were infected with JEV at an MOI of 5, followed by treatment with either 0.5 μM Ag2037 or an equivalent volume of DMSO at 1.5 hpi. The levels of intracellular viral RNA (I) and viral titers in the supernatant (J) were determined at 6, 12, 18, and 24 hpi. (K–L) The effect of hypoxanthine (Hyp) treatment on Ag2037-mediated inhibition of JEV replication. N2a cells infected with JEV were treated with either Ag2037 or DMSO, with or without addition of Hyp at 1.5 hpi. The levels of intracellular viral RNA (K) and viral titers in the supernatant (L) were measured at 18 hpi. Data are presented as mean ± s.e.m. from 3 independent biological replicates (n = 3). Significance was determined using two-tailed unpaired Student’s t-test (B) , one-way (F, H, K and L) or two-way ANOVA (C, E, I and J) (*p < 0.05, **p < 0.01, ***p < 0.001; ****p < 0.0001; ns, not significant).

    Article Snippet: Rabbit anti-GART pAb (A3876), anti-ADSS mAb (A20945), anti-IMPDH2 mAb (A9208), anti-GMPS pAb (A6606), anti-G6PD pAb (A13983), anti-MTHFD1 pAb (A8661), anti-SHMT2 pAb (A1215), anti-MTHFD2 mAb (A22653), and anti-β-Tubulin mAb (A12289) were purchased from ABclonal Technology.

    Techniques: Infection, Expressing, Western Blot, Software, Control, Virus, Quantitative RT-PCR, Plaque Assay, Inhibition, Two Tailed Test

    PSMD14 interacts with IMPDH2 and regulates its protein stability in GBM. (A) Western blot confirming overexpression of exogenous Flag-tagged PSMD14 in U118MG cells. (B) The Co-IP assay of PSMD14 from U118MG cells. (C) Mass spectrometry identification of proteins co-precipitated with PSMD14 in U118MG cells, followed by intersection analysis to identify common candidate interactors. (D) Bioinformatic overlap of the PSMD14 interactome identified by mass spectrometry with known interactions in the BioGRID database. (E) Network analysis of high-confidence interacting proteins with PSMD14. (F) Western blotting analysis to detect changes in IMPDH2 expression after knockdown of PSMD14. (G) The Co-IP assay confirming the interaction between PSMD14 and IMPDH2 in GBM cells. (H) Structural docking model of PSMD14 and IMPDH2 showing the predicted binding interface rendered with PyMOL (PSMD14 in pink, IMPDH2 in purple). (I) Schematic diagram of the PSMD14 and IMPDH2 domain structures and the design of truncation mutants. (J) The Co-IP analysis using PSMD14 and IMPDH2 truncation mutants, identifying the regions required for their interaction. (K) Cycloheximide (CHX) chase assay in control and PSMD14-knockdown GBM cells, evaluating IMPDH2 protein stability over time. (L) Quantification of IMPDH2 protein half-life from CHX chase assays. Data are presented as mean ± SD (n = 3). *P < 0.05 (one-way ANOVA).

    Journal: Theranostics

    Article Title: Coupling proteostasis and de novo purine biosynthesis of PSMD14 fuels glioblastoma progression and chemoresistance

    doi: 10.7150/thno.124409

    Figure Lengend Snippet: PSMD14 interacts with IMPDH2 and regulates its protein stability in GBM. (A) Western blot confirming overexpression of exogenous Flag-tagged PSMD14 in U118MG cells. (B) The Co-IP assay of PSMD14 from U118MG cells. (C) Mass spectrometry identification of proteins co-precipitated with PSMD14 in U118MG cells, followed by intersection analysis to identify common candidate interactors. (D) Bioinformatic overlap of the PSMD14 interactome identified by mass spectrometry with known interactions in the BioGRID database. (E) Network analysis of high-confidence interacting proteins with PSMD14. (F) Western blotting analysis to detect changes in IMPDH2 expression after knockdown of PSMD14. (G) The Co-IP assay confirming the interaction between PSMD14 and IMPDH2 in GBM cells. (H) Structural docking model of PSMD14 and IMPDH2 showing the predicted binding interface rendered with PyMOL (PSMD14 in pink, IMPDH2 in purple). (I) Schematic diagram of the PSMD14 and IMPDH2 domain structures and the design of truncation mutants. (J) The Co-IP analysis using PSMD14 and IMPDH2 truncation mutants, identifying the regions required for their interaction. (K) Cycloheximide (CHX) chase assay in control and PSMD14-knockdown GBM cells, evaluating IMPDH2 protein stability over time. (L) Quantification of IMPDH2 protein half-life from CHX chase assays. Data are presented as mean ± SD (n = 3). *P < 0.05 (one-way ANOVA).

    Article Snippet: Membranes were blocked in 5% (w/v) non-fat milk in TBST for 1 h, then incubated overnight at 4 °C with primary antibodies targeting PSMD14 (4197S; 1:1,000; Cell Signaling Technology), IMPDH2 (36281S; 1:1,000; Cell Signaling Technology), β-Actin (4970S; 1:1,000; Cell Signaling Technology), ubiquitin (20326S; 1:1,000; Cell Signaling Technology), K48-linkage-specific polyubiquitin (8081S; 1:1,000; Cell Signaling Technology), K63-linkage-specific polyubiquitin (5621S; 1:1,000; Cell Signaling Technology), His-Tag (12698T; 1:1000; Cell Signaling Technology), HA-Tag (3724S; 1:1000; Cell Signaling Technology) and Myc-Tag (2276S; 1:1,000; Cell Signaling Technology).

    Techniques: Western Blot, Over Expression, Co-Immunoprecipitation Assay, Mass Spectrometry, Expressing, Knockdown, Binding Assay, Control

    PSMD14 regulates IMPDH2 stability through selective removal of K48-linked ubiquitin chains. (A-C) Western blotting analysis of ubiquitin conjugates in LN229 cells with PSMD14 knockdown. (D-F) Western blotting analysis of ubiquitin conjugates in GBM#P3 cells with PSMD14 knockdown. (G, H) Ubiquitination status of IMPDH2 upon PSMD14 inhibition. GBM cells were treated with the PSMD14 inhibitor thiolutin in the presence of MG132. (I-L) Domain mapping of PSMD14's deubiquitinating function. GBM cells were co-transfected with wild-type PSMD14 or N-terminal truncation mutants along with plasmids encoding Myc-tagged ubiquitin.

    Journal: Theranostics

    Article Title: Coupling proteostasis and de novo purine biosynthesis of PSMD14 fuels glioblastoma progression and chemoresistance

    doi: 10.7150/thno.124409

    Figure Lengend Snippet: PSMD14 regulates IMPDH2 stability through selective removal of K48-linked ubiquitin chains. (A-C) Western blotting analysis of ubiquitin conjugates in LN229 cells with PSMD14 knockdown. (D-F) Western blotting analysis of ubiquitin conjugates in GBM#P3 cells with PSMD14 knockdown. (G, H) Ubiquitination status of IMPDH2 upon PSMD14 inhibition. GBM cells were treated with the PSMD14 inhibitor thiolutin in the presence of MG132. (I-L) Domain mapping of PSMD14's deubiquitinating function. GBM cells were co-transfected with wild-type PSMD14 or N-terminal truncation mutants along with plasmids encoding Myc-tagged ubiquitin.

    Article Snippet: Membranes were blocked in 5% (w/v) non-fat milk in TBST for 1 h, then incubated overnight at 4 °C with primary antibodies targeting PSMD14 (4197S; 1:1,000; Cell Signaling Technology), IMPDH2 (36281S; 1:1,000; Cell Signaling Technology), β-Actin (4970S; 1:1,000; Cell Signaling Technology), ubiquitin (20326S; 1:1,000; Cell Signaling Technology), K48-linkage-specific polyubiquitin (8081S; 1:1,000; Cell Signaling Technology), K63-linkage-specific polyubiquitin (5621S; 1:1,000; Cell Signaling Technology), His-Tag (12698T; 1:1000; Cell Signaling Technology), HA-Tag (3724S; 1:1000; Cell Signaling Technology) and Myc-Tag (2276S; 1:1,000; Cell Signaling Technology).

    Techniques: Ubiquitin Proteomics, Western Blot, Knockdown, Inhibition, Transfection

    Loss of PSMD14 disrupts nucleotide metabolism, induces DNA damage and impairs mitochondrial function in GBM cells. (A) Schematic depiction of metabolic pathways regulated by IMPDH2 in GBM cells. (B-D) Relative levels of metabolites in GBM cells expressing siIMPDH2 or shPSMD14. Data are presented as mean ± SD (n = 3). **P < 0.01, ***P < 0.001 (one-way ANOVA). (E) ICC images showing accumulation of γ-H2AX in GBM cells upon PSMD14 knockdown. Scale bar = 30 μm. (F) ICC images indicating nucleolar stress in PSMD14-knockdown GBM cells. Nucleoli are labeled by nucleostemin. Scale bar = 30 μm. (G) ICC images of mitochondria in PSMD14-knockdown cells. Scale bar = 30 μm. (H) Seahorse extracellular flux analysis of mitochondrial respiration in control and PSMD14-knockdown GBM cells. Data are presented as mean ± SD (n = 3). *P < 0.05, **P < 0.01 (Independent-sample Student-T test). (I) Flow cytometric analysis of mitochondrial membrane potential in control and PSMD14-knockdown cells. Data are presented as mean ± SD (n = 3). *P < 0.05, **P < 0.01 (Independent-sample Student-T test). (J-K) ICC images of JC-1 staining showing changes in mitochondrial membrane potential. Scale bar = 30 μm.

    Journal: Theranostics

    Article Title: Coupling proteostasis and de novo purine biosynthesis of PSMD14 fuels glioblastoma progression and chemoresistance

    doi: 10.7150/thno.124409

    Figure Lengend Snippet: Loss of PSMD14 disrupts nucleotide metabolism, induces DNA damage and impairs mitochondrial function in GBM cells. (A) Schematic depiction of metabolic pathways regulated by IMPDH2 in GBM cells. (B-D) Relative levels of metabolites in GBM cells expressing siIMPDH2 or shPSMD14. Data are presented as mean ± SD (n = 3). **P < 0.01, ***P < 0.001 (one-way ANOVA). (E) ICC images showing accumulation of γ-H2AX in GBM cells upon PSMD14 knockdown. Scale bar = 30 μm. (F) ICC images indicating nucleolar stress in PSMD14-knockdown GBM cells. Nucleoli are labeled by nucleostemin. Scale bar = 30 μm. (G) ICC images of mitochondria in PSMD14-knockdown cells. Scale bar = 30 μm. (H) Seahorse extracellular flux analysis of mitochondrial respiration in control and PSMD14-knockdown GBM cells. Data are presented as mean ± SD (n = 3). *P < 0.05, **P < 0.01 (Independent-sample Student-T test). (I) Flow cytometric analysis of mitochondrial membrane potential in control and PSMD14-knockdown cells. Data are presented as mean ± SD (n = 3). *P < 0.05, **P < 0.01 (Independent-sample Student-T test). (J-K) ICC images of JC-1 staining showing changes in mitochondrial membrane potential. Scale bar = 30 μm.

    Article Snippet: Membranes were blocked in 5% (w/v) non-fat milk in TBST for 1 h, then incubated overnight at 4 °C with primary antibodies targeting PSMD14 (4197S; 1:1,000; Cell Signaling Technology), IMPDH2 (36281S; 1:1,000; Cell Signaling Technology), β-Actin (4970S; 1:1,000; Cell Signaling Technology), ubiquitin (20326S; 1:1,000; Cell Signaling Technology), K48-linkage-specific polyubiquitin (8081S; 1:1,000; Cell Signaling Technology), K63-linkage-specific polyubiquitin (5621S; 1:1,000; Cell Signaling Technology), His-Tag (12698T; 1:1000; Cell Signaling Technology), HA-Tag (3724S; 1:1000; Cell Signaling Technology) and Myc-Tag (2276S; 1:1,000; Cell Signaling Technology).

    Techniques: Expressing, Knockdown, Labeling, Control, Membrane, Staining

    Pharmacological inhibition of PSMD14 with thiolutin reduces tumor burden and synergizes with temozolomide in GBM. (A) IC50 curves of Thiolutin in the GBM#P3, U118, and LN229 cell lines. (B) Time-course of cell viability in GBM cells with ectopic PSMD14 overexpression or empty vector control, in the presence of DMSO or thiolutin. (C) Quantification of cell viability (OD values) on Day 4 (n = 3). ***P <0.001 (one-way ANOVA). (D) Representative bioluminescence images of intracranial GBM#P3 xenograft-bearing mice at Days 7 and 28 post-implantation. Mice were treated with vehicle or thiolutin, with or without PSMD14 overexpression in the implanted cells. (E) Quantification of total photon flux from intracranial tumors at Day 28. Data are presented as mean ± SD (n = 5 per group). ***P <0.001 (one-way ANOVA). (F) Kaplan-Meier survival analysis of mice bearing intracranial tumors under the indicated treatments (n = 10 per group). (G) Representative HE is staining of brain sections from tumor-bearing mice across treatment groups. Scale bar = 2.5 mm. (H) IHC staining for PSMD14 and IMPDH2 in brain tumor sections from the orthotopic xenografts. Scale bar = 100 μm. (I) Representative IF images of tumor tissues from the xenograft models. Scale bar = 100 μm. (J) Cell viability curves for GBM cells treated for 4 days with vehicle (DMSO), TMZ, thiolutin, or the combination of TMZ + thiolutin. (K) Quantification of cell viability (OD) at Day 4. Data are presented as mean ± SD (n = 3). ***P <0.001 (one-way ANOVA). (L) Bioluminescent imaging of intracranial tumors in mice treated with vehicle, TMZ, or TMZ + thiolutin on Days 7 and 28 post-implantation. (M) Quantification of photon flux at Day 28. Data are presented as mean ± SD (n = 5 per group). *P < 0.05, **P < 0.01, ***P < 0.001 (one-way ANOVA). (N) Representative IHC images of PSMD14 and IMPDH2 in tumor tissues from mice treated with TMZ alone or TMZ + thiolutin. Scale bar = 100 μm.

    Journal: Theranostics

    Article Title: Coupling proteostasis and de novo purine biosynthesis of PSMD14 fuels glioblastoma progression and chemoresistance

    doi: 10.7150/thno.124409

    Figure Lengend Snippet: Pharmacological inhibition of PSMD14 with thiolutin reduces tumor burden and synergizes with temozolomide in GBM. (A) IC50 curves of Thiolutin in the GBM#P3, U118, and LN229 cell lines. (B) Time-course of cell viability in GBM cells with ectopic PSMD14 overexpression or empty vector control, in the presence of DMSO or thiolutin. (C) Quantification of cell viability (OD values) on Day 4 (n = 3). ***P <0.001 (one-way ANOVA). (D) Representative bioluminescence images of intracranial GBM#P3 xenograft-bearing mice at Days 7 and 28 post-implantation. Mice were treated with vehicle or thiolutin, with or without PSMD14 overexpression in the implanted cells. (E) Quantification of total photon flux from intracranial tumors at Day 28. Data are presented as mean ± SD (n = 5 per group). ***P <0.001 (one-way ANOVA). (F) Kaplan-Meier survival analysis of mice bearing intracranial tumors under the indicated treatments (n = 10 per group). (G) Representative HE is staining of brain sections from tumor-bearing mice across treatment groups. Scale bar = 2.5 mm. (H) IHC staining for PSMD14 and IMPDH2 in brain tumor sections from the orthotopic xenografts. Scale bar = 100 μm. (I) Representative IF images of tumor tissues from the xenograft models. Scale bar = 100 μm. (J) Cell viability curves for GBM cells treated for 4 days with vehicle (DMSO), TMZ, thiolutin, or the combination of TMZ + thiolutin. (K) Quantification of cell viability (OD) at Day 4. Data are presented as mean ± SD (n = 3). ***P <0.001 (one-way ANOVA). (L) Bioluminescent imaging of intracranial tumors in mice treated with vehicle, TMZ, or TMZ + thiolutin on Days 7 and 28 post-implantation. (M) Quantification of photon flux at Day 28. Data are presented as mean ± SD (n = 5 per group). *P < 0.05, **P < 0.01, ***P < 0.001 (one-way ANOVA). (N) Representative IHC images of PSMD14 and IMPDH2 in tumor tissues from mice treated with TMZ alone or TMZ + thiolutin. Scale bar = 100 μm.

    Article Snippet: Membranes were blocked in 5% (w/v) non-fat milk in TBST for 1 h, then incubated overnight at 4 °C with primary antibodies targeting PSMD14 (4197S; 1:1,000; Cell Signaling Technology), IMPDH2 (36281S; 1:1,000; Cell Signaling Technology), β-Actin (4970S; 1:1,000; Cell Signaling Technology), ubiquitin (20326S; 1:1,000; Cell Signaling Technology), K48-linkage-specific polyubiquitin (8081S; 1:1,000; Cell Signaling Technology), K63-linkage-specific polyubiquitin (5621S; 1:1,000; Cell Signaling Technology), His-Tag (12698T; 1:1000; Cell Signaling Technology), HA-Tag (3724S; 1:1000; Cell Signaling Technology) and Myc-Tag (2276S; 1:1,000; Cell Signaling Technology).

    Techniques: Inhibition, Over Expression, Plasmid Preparation, Control, Staining, Immunohistochemistry, Imaging