p stat1 antibody py701 4a Search Results


stat1 py701 antibody, anti-human/mouse, reafinity  (Miltenyi Biotec)
93
Miltenyi Biotec stat1 py701 antibody, anti-human/mouse, reafinity
Stat1 Py701 Antibody, Anti Human/Mouse, Reafinity, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 93 stars, based on 1 article reviews
stat1 py701 antibody, anti-human/mouse, reafinity - by Bioz Stars, 2026-11
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bv421-anti-stat1 py701  (Becton Dickinson)
90
Becton Dickinson bv421-anti-stat1 py701
Lentivirus mediated ADA2 gene transfer restores ADA2 protein expression, enzyme activity and rescues immunophenotype in monocyte derived macrophages (MDM) from DADA2 patients (A) . Representative flow cytometric histogram showing ADA2 expression (MFI, median fluorescence intensity) in MDM obtained from patients with DADA2 and healthy controls. (B) EFS-GFP-ADA2 transduction of macrophage cells derived from patients with DADA2 resulted in restoration of ADA2 protein expression assessed by flow cytometry compared to no change in protein expression in EFS-GFP alone treated cells. (C) ADA2 enzyme activity assessed by an automated spectrophotometric assay also recovered in the culture supernatant of EFS-GFP-ADA2 transduced MDM from DADA2 patients compared to supernatants derived from EFS-GFP transduced cells. (D, E) EFS-GFP-ADA2 transduction of MDM derived from patients also led to a reduction in levels of TNF-α cytokine production in culture supernatants compared to EFS-GFP alone treated cells. Cumulative results and individual changes for each sample assessed are shown. (F, G) There was also suppression of IFN-γ release expression in culture supernatants and downregulation of <t>p-STAT1</t> expression in EFS-GFP-ADA2 transduced MDM compared to EFS-GFP treated cells. (H) There was significant improvement in CD62E expression (MFI) on HUVEC incubated with culture supernatants from EFS-GFP-ADA2 transduced MDM from patients with DADA2 compared to co incubation with supernatants from EFS-GFP treated MDM. Dotted line represents levels observed in healthy control samples. MDM, monocyte derived macrophages; MFI, median fluorescence intensity; TNFα, tumour necrosis factor-α; IL, interleukin; EFS, elongation factor 1α short; IFN, interferon; GFP, green fluorescent protein; HUVEC, human umbilical vein endothelial cells; INF, interferon; UT, untransduced: ADA2, adenosine deaminase 2.
Bv421 Anti Stat1 Py701, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 90 stars, based on 1 article reviews
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py701 stat1  (Cell Signaling Technology Inc)
97
Cell Signaling Technology Inc py701 stat1
FIGURE 3. USP39 promotes type I IFN antiviral signaling. (A) 293T cells were transfected with either control siRNA or siUSP39, together with ISRE- Luc and Renilla luciferase reporter plasmid for 48 h. The luciferase activity was measured after further 12 h IFN-a (1000 IU/ml) treatment. (B) 293T cells were transfected with either vector or Flag-USP39, together with ISRE-Luc and Renilla for 48 h. The luciferase activity was measured after further 12 h of IFN-a (1000 IU/ml) treatment. (C) 293T cells were transfected with either control siRNA or siUSP39 for 48 h and treated with IFN-a (1000 IU/ml) for 4 and 8 h, and mRNA levels of IFIT1 and ISG54 were analyzed by real-time PCR. (D) 293T cells were transfected with either vector or Flag-USP39 for 48 h and treated with IFN-a (1000 IU/ml) for 4 and 8 h. The mRNA levels of IFIT1 and ISG54 were analyzed by real-time PCR. (E) U3A <t>(STAT1</t> deficient) or its parental cells, HT1080, were transfected with either control siRNA or siUSP39. After 48 h of transfection, cells were infected with VSV-GFP (MOI = 1.0) for 12 h. The knockdown efficiency of USP39 was analyzed by Western blot. Viral levels were analyzed by flow cytometry. Data are from three experiments and are mean and SD of three independent replicates. (A–E, right panels) or representative of three independent experiments (E left panel). *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001. A p value .0.05 was considered ns.
Py701 Stat1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/p+stat1+antibody+py701+4a/Phospho-Stat1+(Tyr701)+Rabbit+mAb/pm33127822-87-39-44
Average 97 stars, based on 1 article reviews
py701 stat1 - by Bioz Stars, 2026-11
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p stat1 antibody py701 4a  (Cell Signaling Technology Inc)
97
Cell Signaling Technology Inc p stat1 antibody py701 4a
FIGURE 3. USP39 promotes type I IFN antiviral signaling. (A) 293T cells were transfected with either control siRNA or siUSP39, together with ISRE- Luc and Renilla luciferase reporter plasmid for 48 h. The luciferase activity was measured after further 12 h IFN-a (1000 IU/ml) treatment. (B) 293T cells were transfected with either vector or Flag-USP39, together with ISRE-Luc and Renilla for 48 h. The luciferase activity was measured after further 12 h of IFN-a (1000 IU/ml) treatment. (C) 293T cells were transfected with either control siRNA or siUSP39 for 48 h and treated with IFN-a (1000 IU/ml) for 4 and 8 h, and mRNA levels of IFIT1 and ISG54 were analyzed by real-time PCR. (D) 293T cells were transfected with either vector or Flag-USP39 for 48 h and treated with IFN-a (1000 IU/ml) for 4 and 8 h. The mRNA levels of IFIT1 and ISG54 were analyzed by real-time PCR. (E) U3A <t>(STAT1</t> deficient) or its parental cells, HT1080, were transfected with either control siRNA or siUSP39. After 48 h of transfection, cells were infected with VSV-GFP (MOI = 1.0) for 12 h. The knockdown efficiency of USP39 was analyzed by Western blot. Viral levels were analyzed by flow cytometry. Data are from three experiments and are mean and SD of three independent replicates. (A–E, right panels) or representative of three independent experiments (E left panel). *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001. A p value .0.05 was considered ns.
P Stat1 Antibody Py701 4a, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 97 stars, based on 1 article reviews
p stat1 antibody py701 4a - by Bioz Stars, 2026-11
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anti-phospho-stat1  (Becton Dickinson)
90
Becton Dickinson anti-phospho-stat1
FIGURE 3. USP39 promotes type I IFN antiviral signaling. (A) 293T cells were transfected with either control siRNA or siUSP39, together with ISRE- Luc and Renilla luciferase reporter plasmid for 48 h. The luciferase activity was measured after further 12 h IFN-a (1000 IU/ml) treatment. (B) 293T cells were transfected with either vector or Flag-USP39, together with ISRE-Luc and Renilla for 48 h. The luciferase activity was measured after further 12 h of IFN-a (1000 IU/ml) treatment. (C) 293T cells were transfected with either control siRNA or siUSP39 for 48 h and treated with IFN-a (1000 IU/ml) for 4 and 8 h, and mRNA levels of IFIT1 and ISG54 were analyzed by real-time PCR. (D) 293T cells were transfected with either vector or Flag-USP39 for 48 h and treated with IFN-a (1000 IU/ml) for 4 and 8 h. The mRNA levels of IFIT1 and ISG54 were analyzed by real-time PCR. (E) U3A <t>(STAT1</t> deficient) or its parental cells, HT1080, were transfected with either control siRNA or siUSP39. After 48 h of transfection, cells were infected with VSV-GFP (MOI = 1.0) for 12 h. The knockdown efficiency of USP39 was analyzed by Western blot. Viral levels were analyzed by flow cytometry. Data are from three experiments and are mean and SD of three independent replicates. (A–E, right panels) or representative of three independent experiments (E left panel). *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001. A p value .0.05 was considered ns.
Anti Phospho Stat1, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 90 stars, based on 1 article reviews
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anti human cd14 efluor 450 plus anti human pstat1 py701 alexa fluor 647  (Cell Signaling Technology Inc)
93
Cell Signaling Technology Inc anti human cd14 efluor 450 plus anti human pstat1 py701 alexa fluor 647
FIGURE 3. USP39 promotes type I IFN antiviral signaling. (A) 293T cells were transfected with either control siRNA or siUSP39, together with ISRE- Luc and Renilla luciferase reporter plasmid for 48 h. The luciferase activity was measured after further 12 h IFN-a (1000 IU/ml) treatment. (B) 293T cells were transfected with either vector or Flag-USP39, together with ISRE-Luc and Renilla for 48 h. The luciferase activity was measured after further 12 h of IFN-a (1000 IU/ml) treatment. (C) 293T cells were transfected with either control siRNA or siUSP39 for 48 h and treated with IFN-a (1000 IU/ml) for 4 and 8 h, and mRNA levels of IFIT1 and ISG54 were analyzed by real-time PCR. (D) 293T cells were transfected with either vector or Flag-USP39 for 48 h and treated with IFN-a (1000 IU/ml) for 4 and 8 h. The mRNA levels of IFIT1 and ISG54 were analyzed by real-time PCR. (E) U3A <t>(STAT1</t> deficient) or its parental cells, HT1080, were transfected with either control siRNA or siUSP39. After 48 h of transfection, cells were infected with VSV-GFP (MOI = 1.0) for 12 h. The knockdown efficiency of USP39 was analyzed by Western blot. Viral levels were analyzed by flow cytometry. Data are from three experiments and are mean and SD of three independent replicates. (A–E, right panels) or representative of three independent experiments (E left panel). *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001. A p value .0.05 was considered ns.
Anti Human Cd14 Efluor 450 Plus Anti Human Pstat1 Py701 Alexa Fluor 647, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 93 stars, based on 1 article reviews
anti human cd14 efluor 450 plus anti human pstat1 py701 alexa fluor 647 - by Bioz Stars, 2026-11
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cst 9145s anti phospho stat1  (Cell Signaling Technology Inc)
98
Cell Signaling Technology Inc cst 9145s anti phospho stat1
FIGURE 3. USP39 promotes type I IFN antiviral signaling. (A) 293T cells were transfected with either control siRNA or siUSP39, together with ISRE- Luc and Renilla luciferase reporter plasmid for 48 h. The luciferase activity was measured after further 12 h IFN-a (1000 IU/ml) treatment. (B) 293T cells were transfected with either vector or Flag-USP39, together with ISRE-Luc and Renilla for 48 h. The luciferase activity was measured after further 12 h of IFN-a (1000 IU/ml) treatment. (C) 293T cells were transfected with either control siRNA or siUSP39 for 48 h and treated with IFN-a (1000 IU/ml) for 4 and 8 h, and mRNA levels of IFIT1 and ISG54 were analyzed by real-time PCR. (D) 293T cells were transfected with either vector or Flag-USP39 for 48 h and treated with IFN-a (1000 IU/ml) for 4 and 8 h. The mRNA levels of IFIT1 and ISG54 were analyzed by real-time PCR. (E) U3A <t>(STAT1</t> deficient) or its parental cells, HT1080, were transfected with either control siRNA or siUSP39. After 48 h of transfection, cells were infected with VSV-GFP (MOI = 1.0) for 12 h. The knockdown efficiency of USP39 was analyzed by Western blot. Viral levels were analyzed by flow cytometry. Data are from three experiments and are mean and SD of three independent replicates. (A–E, right panels) or representative of three independent experiments (E left panel). *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001. A p value .0.05 was considered ns.
Cst 9145s Anti Phospho Stat1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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phospho stat1 py701 4 a mouse monoclonal igg2aĸ targeting  (Santa Cruz Biotechnology)
94
Santa Cruz Biotechnology phospho stat1 py701 4 a mouse monoclonal igg2aĸ targeting
FIGURE 3. USP39 promotes type I IFN antiviral signaling. (A) 293T cells were transfected with either control siRNA or siUSP39, together with ISRE- Luc and Renilla luciferase reporter plasmid for 48 h. The luciferase activity was measured after further 12 h IFN-a (1000 IU/ml) treatment. (B) 293T cells were transfected with either vector or Flag-USP39, together with ISRE-Luc and Renilla for 48 h. The luciferase activity was measured after further 12 h of IFN-a (1000 IU/ml) treatment. (C) 293T cells were transfected with either control siRNA or siUSP39 for 48 h and treated with IFN-a (1000 IU/ml) for 4 and 8 h, and mRNA levels of IFIT1 and ISG54 were analyzed by real-time PCR. (D) 293T cells were transfected with either vector or Flag-USP39 for 48 h and treated with IFN-a (1000 IU/ml) for 4 and 8 h. The mRNA levels of IFIT1 and ISG54 were analyzed by real-time PCR. (E) U3A <t>(STAT1</t> deficient) or its parental cells, HT1080, were transfected with either control siRNA or siUSP39. After 48 h of transfection, cells were infected with VSV-GFP (MOI = 1.0) for 12 h. The knockdown efficiency of USP39 was analyzed by Western blot. Viral levels were analyzed by flow cytometry. Data are from three experiments and are mean and SD of three independent replicates. (A–E, right panels) or representative of three independent experiments (E left panel). *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001. A p value .0.05 was considered ns.
Phospho Stat1 Py701 4 A Mouse Monoclonal Igg2aĸ Targeting, supplied by Santa Cruz Biotechnology, 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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anti stat1 py701 antibodies  (Santa Cruz Biotechnology)
96
Santa Cruz Biotechnology anti stat1 py701 antibodies
FIGURE 3. USP39 promotes type I IFN antiviral signaling. (A) 293T cells were transfected with either control siRNA or siUSP39, together with ISRE- Luc and Renilla luciferase reporter plasmid for 48 h. The luciferase activity was measured after further 12 h IFN-a (1000 IU/ml) treatment. (B) 293T cells were transfected with either vector or Flag-USP39, together with ISRE-Luc and Renilla for 48 h. The luciferase activity was measured after further 12 h of IFN-a (1000 IU/ml) treatment. (C) 293T cells were transfected with either control siRNA or siUSP39 for 48 h and treated with IFN-a (1000 IU/ml) for 4 and 8 h, and mRNA levels of IFIT1 and ISG54 were analyzed by real-time PCR. (D) 293T cells were transfected with either vector or Flag-USP39 for 48 h and treated with IFN-a (1000 IU/ml) for 4 and 8 h. The mRNA levels of IFIT1 and ISG54 were analyzed by real-time PCR. (E) U3A <t>(STAT1</t> deficient) or its parental cells, HT1080, were transfected with either control siRNA or siUSP39. After 48 h of transfection, cells were infected with VSV-GFP (MOI = 1.0) for 12 h. The knockdown efficiency of USP39 was analyzed by Western blot. Viral levels were analyzed by flow cytometry. Data are from three experiments and are mean and SD of three independent replicates. (A–E, right panels) or representative of three independent experiments (E left panel). *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001. A p value .0.05 was considered ns.
Anti Stat1 Py701 Antibodies, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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anti stat1 py701 antibodies - by Bioz Stars, 2026-11
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phospho stat1  (Cell Signaling Technology Inc)
96
Cell Signaling Technology Inc phospho stat1
FIGURE 3. USP39 promotes type I IFN antiviral signaling. (A) 293T cells were transfected with either control siRNA or siUSP39, together with ISRE- Luc and Renilla luciferase reporter plasmid for 48 h. The luciferase activity was measured after further 12 h IFN-a (1000 IU/ml) treatment. (B) 293T cells were transfected with either vector or Flag-USP39, together with ISRE-Luc and Renilla for 48 h. The luciferase activity was measured after further 12 h of IFN-a (1000 IU/ml) treatment. (C) 293T cells were transfected with either control siRNA or siUSP39 for 48 h and treated with IFN-a (1000 IU/ml) for 4 and 8 h, and mRNA levels of IFIT1 and ISG54 were analyzed by real-time PCR. (D) 293T cells were transfected with either vector or Flag-USP39 for 48 h and treated with IFN-a (1000 IU/ml) for 4 and 8 h. The mRNA levels of IFIT1 and ISG54 were analyzed by real-time PCR. (E) U3A <t>(STAT1</t> deficient) or its parental cells, HT1080, were transfected with either control siRNA or siUSP39. After 48 h of transfection, cells were infected with VSV-GFP (MOI = 1.0) for 12 h. The knockdown efficiency of USP39 was analyzed by Western blot. Viral levels were analyzed by flow cytometry. Data are from three experiments and are mean and SD of three independent replicates. (A–E, right panels) or representative of three independent experiments (E left panel). *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001. A p value .0.05 was considered ns.
Phospho Stat1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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antibodies rabbit anti py701 stat1  (Santa Cruz Biotechnology)
94
Santa Cruz Biotechnology antibodies rabbit anti py701 stat1
FIGURE 3. USP39 promotes type I IFN antiviral signaling. (A) 293T cells were transfected with either control siRNA or siUSP39, together with ISRE- Luc and Renilla luciferase reporter plasmid for 48 h. The luciferase activity was measured after further 12 h IFN-a (1000 IU/ml) treatment. (B) 293T cells were transfected with either vector or Flag-USP39, together with ISRE-Luc and Renilla for 48 h. The luciferase activity was measured after further 12 h of IFN-a (1000 IU/ml) treatment. (C) 293T cells were transfected with either control siRNA or siUSP39 for 48 h and treated with IFN-a (1000 IU/ml) for 4 and 8 h, and mRNA levels of IFIT1 and ISG54 were analyzed by real-time PCR. (D) 293T cells were transfected with either vector or Flag-USP39 for 48 h and treated with IFN-a (1000 IU/ml) for 4 and 8 h. The mRNA levels of IFIT1 and ISG54 were analyzed by real-time PCR. (E) U3A <t>(STAT1</t> deficient) or its parental cells, HT1080, were transfected with either control siRNA or siUSP39. After 48 h of transfection, cells were infected with VSV-GFP (MOI = 1.0) for 12 h. The knockdown efficiency of USP39 was analyzed by Western blot. Viral levels were analyzed by flow cytometry. Data are from three experiments and are mean and SD of three independent replicates. (A–E, right panels) or representative of three independent experiments (E left panel). *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001. A p value .0.05 was considered ns.
Antibodies Rabbit Anti Py701 Stat1, supplied by Santa Cruz Biotechnology, 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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antibodies rabbit anti py701 stat1 - by Bioz Stars, 2026-11
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monoclonal rabbit anti py701 stat1  (Cell Signaling Technology Inc)
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Cell Signaling Technology Inc monoclonal rabbit anti py701 stat1
FIGURE 3. USP39 promotes type I IFN antiviral signaling. (A) 293T cells were transfected with either control siRNA or siUSP39, together with ISRE- Luc and Renilla luciferase reporter plasmid for 48 h. The luciferase activity was measured after further 12 h IFN-a (1000 IU/ml) treatment. (B) 293T cells were transfected with either vector or Flag-USP39, together with ISRE-Luc and Renilla for 48 h. The luciferase activity was measured after further 12 h of IFN-a (1000 IU/ml) treatment. (C) 293T cells were transfected with either control siRNA or siUSP39 for 48 h and treated with IFN-a (1000 IU/ml) for 4 and 8 h, and mRNA levels of IFIT1 and ISG54 were analyzed by real-time PCR. (D) 293T cells were transfected with either vector or Flag-USP39 for 48 h and treated with IFN-a (1000 IU/ml) for 4 and 8 h. The mRNA levels of IFIT1 and ISG54 were analyzed by real-time PCR. (E) U3A <t>(STAT1</t> deficient) or its parental cells, HT1080, were transfected with either control siRNA or siUSP39. After 48 h of transfection, cells were infected with VSV-GFP (MOI = 1.0) for 12 h. The knockdown efficiency of USP39 was analyzed by Western blot. Viral levels were analyzed by flow cytometry. Data are from three experiments and are mean and SD of three independent replicates. (A–E, right panels) or representative of three independent experiments (E left panel). *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001. A p value .0.05 was considered ns.
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Image Search Results


Lentivirus mediated ADA2 gene transfer restores ADA2 protein expression, enzyme activity and rescues immunophenotype in monocyte derived macrophages (MDM) from DADA2 patients (A) . Representative flow cytometric histogram showing ADA2 expression (MFI, median fluorescence intensity) in MDM obtained from patients with DADA2 and healthy controls. (B) EFS-GFP-ADA2 transduction of macrophage cells derived from patients with DADA2 resulted in restoration of ADA2 protein expression assessed by flow cytometry compared to no change in protein expression in EFS-GFP alone treated cells. (C) ADA2 enzyme activity assessed by an automated spectrophotometric assay also recovered in the culture supernatant of EFS-GFP-ADA2 transduced MDM from DADA2 patients compared to supernatants derived from EFS-GFP transduced cells. (D, E) EFS-GFP-ADA2 transduction of MDM derived from patients also led to a reduction in levels of TNF-α cytokine production in culture supernatants compared to EFS-GFP alone treated cells. Cumulative results and individual changes for each sample assessed are shown. (F, G) There was also suppression of IFN-γ release expression in culture supernatants and downregulation of p-STAT1 expression in EFS-GFP-ADA2 transduced MDM compared to EFS-GFP treated cells. (H) There was significant improvement in CD62E expression (MFI) on HUVEC incubated with culture supernatants from EFS-GFP-ADA2 transduced MDM from patients with DADA2 compared to co incubation with supernatants from EFS-GFP treated MDM. Dotted line represents levels observed in healthy control samples. MDM, monocyte derived macrophages; MFI, median fluorescence intensity; TNFα, tumour necrosis factor-α; IL, interleukin; EFS, elongation factor 1α short; IFN, interferon; GFP, green fluorescent protein; HUVEC, human umbilical vein endothelial cells; INF, interferon; UT, untransduced: ADA2, adenosine deaminase 2.

Journal: Frontiers in Immunology

Article Title: Lentiviral Mediated ADA2 Gene Transfer Corrects the Defects Associated With Deficiency of Adenosine Deaminase Type 2

doi: 10.3389/fimmu.2022.852830

Figure Lengend Snippet: Lentivirus mediated ADA2 gene transfer restores ADA2 protein expression, enzyme activity and rescues immunophenotype in monocyte derived macrophages (MDM) from DADA2 patients (A) . Representative flow cytometric histogram showing ADA2 expression (MFI, median fluorescence intensity) in MDM obtained from patients with DADA2 and healthy controls. (B) EFS-GFP-ADA2 transduction of macrophage cells derived from patients with DADA2 resulted in restoration of ADA2 protein expression assessed by flow cytometry compared to no change in protein expression in EFS-GFP alone treated cells. (C) ADA2 enzyme activity assessed by an automated spectrophotometric assay also recovered in the culture supernatant of EFS-GFP-ADA2 transduced MDM from DADA2 patients compared to supernatants derived from EFS-GFP transduced cells. (D, E) EFS-GFP-ADA2 transduction of MDM derived from patients also led to a reduction in levels of TNF-α cytokine production in culture supernatants compared to EFS-GFP alone treated cells. Cumulative results and individual changes for each sample assessed are shown. (F, G) There was also suppression of IFN-γ release expression in culture supernatants and downregulation of p-STAT1 expression in EFS-GFP-ADA2 transduced MDM compared to EFS-GFP treated cells. (H) There was significant improvement in CD62E expression (MFI) on HUVEC incubated with culture supernatants from EFS-GFP-ADA2 transduced MDM from patients with DADA2 compared to co incubation with supernatants from EFS-GFP treated MDM. Dotted line represents levels observed in healthy control samples. MDM, monocyte derived macrophages; MFI, median fluorescence intensity; TNFα, tumour necrosis factor-α; IL, interleukin; EFS, elongation factor 1α short; IFN, interferon; GFP, green fluorescent protein; HUVEC, human umbilical vein endothelial cells; INF, interferon; UT, untransduced: ADA2, adenosine deaminase 2.

Article Snippet: Cells were stained with BV421-anti-STAT1 pY701 (BD Bioscience, cat: 562985, 1:50) and cell surface markers APC-CD68 (Miltenyi Biotec, cat: 160-109-462, REA613, 1:30).

Techniques: Expressing, Activity Assay, Derivative Assay, Fluorescence, Transduction, Flow Cytometry, Spectrophotometric Assay, Incubation

FIGURE 3. USP39 promotes type I IFN antiviral signaling. (A) 293T cells were transfected with either control siRNA or siUSP39, together with ISRE- Luc and Renilla luciferase reporter plasmid for 48 h. The luciferase activity was measured after further 12 h IFN-a (1000 IU/ml) treatment. (B) 293T cells were transfected with either vector or Flag-USP39, together with ISRE-Luc and Renilla for 48 h. The luciferase activity was measured after further 12 h of IFN-a (1000 IU/ml) treatment. (C) 293T cells were transfected with either control siRNA or siUSP39 for 48 h and treated with IFN-a (1000 IU/ml) for 4 and 8 h, and mRNA levels of IFIT1 and ISG54 were analyzed by real-time PCR. (D) 293T cells were transfected with either vector or Flag-USP39 for 48 h and treated with IFN-a (1000 IU/ml) for 4 and 8 h. The mRNA levels of IFIT1 and ISG54 were analyzed by real-time PCR. (E) U3A (STAT1 deficient) or its parental cells, HT1080, were transfected with either control siRNA or siUSP39. After 48 h of transfection, cells were infected with VSV-GFP (MOI = 1.0) for 12 h. The knockdown efficiency of USP39 was analyzed by Western blot. Viral levels were analyzed by flow cytometry. Data are from three experiments and are mean and SD of three independent replicates. (A–E, right panels) or representative of three independent experiments (E left panel). *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001. A p value .0.05 was considered ns.

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

Article Title: USP39 Serves as a Deubiquitinase to Stabilize STAT1 and Sustains Type I IFN-Induced Antiviral Immunity.

doi: 10.4049/jimmunol.1901384

Figure Lengend Snippet: FIGURE 3. USP39 promotes type I IFN antiviral signaling. (A) 293T cells were transfected with either control siRNA or siUSP39, together with ISRE- Luc and Renilla luciferase reporter plasmid for 48 h. The luciferase activity was measured after further 12 h IFN-a (1000 IU/ml) treatment. (B) 293T cells were transfected with either vector or Flag-USP39, together with ISRE-Luc and Renilla for 48 h. The luciferase activity was measured after further 12 h of IFN-a (1000 IU/ml) treatment. (C) 293T cells were transfected with either control siRNA or siUSP39 for 48 h and treated with IFN-a (1000 IU/ml) for 4 and 8 h, and mRNA levels of IFIT1 and ISG54 were analyzed by real-time PCR. (D) 293T cells were transfected with either vector or Flag-USP39 for 48 h and treated with IFN-a (1000 IU/ml) for 4 and 8 h. The mRNA levels of IFIT1 and ISG54 were analyzed by real-time PCR. (E) U3A (STAT1 deficient) or its parental cells, HT1080, were transfected with either control siRNA or siUSP39. After 48 h of transfection, cells were infected with VSV-GFP (MOI = 1.0) for 12 h. The knockdown efficiency of USP39 was analyzed by Western blot. Viral levels were analyzed by flow cytometry. Data are from three experiments and are mean and SD of three independent replicates. (A–E, right panels) or representative of three independent experiments (E left panel). *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001. A p value .0.05 was considered ns.

Article Snippet: The following Abs were used: Abs against USP39 (1:2000; ab131244; Abcam, Cambridge, MA), IFNR1 (1:1000; DF6571; Affinity, Cincinnati, OH), IFNR2 (1:1000; sc-271105; Santa Cruz Biotechnology, Santa Cruz, CA), STAT1 (1:1000; catalog no. 9172; Cell Signaling Technology [CST], Danvers, MA), pY701-STAT1 (1:1000; catalog no. 9167; CST), STAT2 (1:1000; catalog no. 72704; CST), pY690-STAT2 (1:1000; catalog no. 88410; CST), JAK1 (1:1000; sc-1677; Santa Cruz Biotechnology), pJAK1 (1:1000; catalog no. 3331s; CST), Tyk2 (1:1000; catalog no. 14193; CST), p-Tyk2 (1:1000; catalog no. 9321; CST), VSV-G (1:5000; sc-66180; Santa Cruz Biotechnology), HA (1:2000; ab9110; Abcam), ubiquitin (1:1000; sc-8017; Santa Cruz Biotechnology), Flag (1:2500; F7425; Sigma-Aldrich), Myc (1:2000; catalog no. 9B11; CST), GAPDH (1:20,000; G9545; Sigma-Aldrich), tubulin (1:5000; 66031-1-Ig; Proteintech, Rosemont, IL).

Techniques: Transfection, Control, Luciferase, Plasmid Preparation, Activity Assay, Real-time Polymerase Chain Reaction, Infection, Knockdown, Western Blot, Cytometry

FIGURE 4. USP39 interacts with STAT1. (A) 293T cells were transfected with either control siRNA or siUSP39, and mRNA levels of type I IFN molecules were measured by real-time PCR. (B) 293T cells transfected with either control siRNA (con siRNA) or siUSP39 were stimulated with IFN-a (1000 IU/ml) for 0, 30, and 60 min. Cell lysates were analyzed by immunoblotting as indicated. (C) 293T cells were transfected with either vector or Flag- USP39. After 48 h, the protein level of STAT1 was measured by Western blot. (D) 293T cells were transfected with Flag-USP39 plasmid. Forty-eight hours posttransfection, the cells were lysed and immunoprecipitated by STAT1 Ab and protein A agarose beads, The USP39 was analyzed using anti-Flag Ab. Data are from three experiments are mean and SD of three independent replicates (A) or representative of three independent experiments (B–D). A p value .0.05 was considered ns.

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

Article Title: USP39 Serves as a Deubiquitinase to Stabilize STAT1 and Sustains Type I IFN-Induced Antiviral Immunity.

doi: 10.4049/jimmunol.1901384

Figure Lengend Snippet: FIGURE 4. USP39 interacts with STAT1. (A) 293T cells were transfected with either control siRNA or siUSP39, and mRNA levels of type I IFN molecules were measured by real-time PCR. (B) 293T cells transfected with either control siRNA (con siRNA) or siUSP39 were stimulated with IFN-a (1000 IU/ml) for 0, 30, and 60 min. Cell lysates were analyzed by immunoblotting as indicated. (C) 293T cells were transfected with either vector or Flag- USP39. After 48 h, the protein level of STAT1 was measured by Western blot. (D) 293T cells were transfected with Flag-USP39 plasmid. Forty-eight hours posttransfection, the cells were lysed and immunoprecipitated by STAT1 Ab and protein A agarose beads, The USP39 was analyzed using anti-Flag Ab. Data are from three experiments are mean and SD of three independent replicates (A) or representative of three independent experiments (B–D). A p value .0.05 was considered ns.

Article Snippet: The following Abs were used: Abs against USP39 (1:2000; ab131244; Abcam, Cambridge, MA), IFNR1 (1:1000; DF6571; Affinity, Cincinnati, OH), IFNR2 (1:1000; sc-271105; Santa Cruz Biotechnology, Santa Cruz, CA), STAT1 (1:1000; catalog no. 9172; Cell Signaling Technology [CST], Danvers, MA), pY701-STAT1 (1:1000; catalog no. 9167; CST), STAT2 (1:1000; catalog no. 72704; CST), pY690-STAT2 (1:1000; catalog no. 88410; CST), JAK1 (1:1000; sc-1677; Santa Cruz Biotechnology), pJAK1 (1:1000; catalog no. 3331s; CST), Tyk2 (1:1000; catalog no. 14193; CST), p-Tyk2 (1:1000; catalog no. 9321; CST), VSV-G (1:5000; sc-66180; Santa Cruz Biotechnology), HA (1:2000; ab9110; Abcam), ubiquitin (1:1000; sc-8017; Santa Cruz Biotechnology), Flag (1:2500; F7425; Sigma-Aldrich), Myc (1:2000; catalog no. 9B11; CST), GAPDH (1:20,000; G9545; Sigma-Aldrich), tubulin (1:5000; 66031-1-Ig; Proteintech, Rosemont, IL).

Techniques: Transfection, Control, Real-time Polymerase Chain Reaction, Western Blot, Plasmid Preparation, Immunoprecipitation

FIGURE 5. USP39 stabilizes STAT1 protein level. (A) 293T cells were transfected with either vector or Flag-USP39 and then treated with CHX (50 mg/ml) for the indicated times. Cell lysates were analyzed by immunoblotting as indicated. (B) 293T cells were transfected with either control siRNA or siUSP39, together with HA-Ub plasmids. STAT1 proteins were immunoprecipitated, and STAT1 ubiquitination levels were analyzed. (C) 293T cells were cotransfected with Flag-STAT1 and HA-Ub or Flag-USP39 expression plasmids, respectively. Forty-eight hours posttransfection, the Flag-STAT1 and Flag- USP39 proteins were purified from 293T cells by anti-Flag beads. Both immunoprecipitated proteins were incubated at 37˚C for 2 h with deubiquitination buffer, followed by immunoblotting as indicated for in vitro deubiquitination assay. (D) Myc-STAT1 proteins were purified from 293T cells cotransfected with Myc-STAT1 and HA-Ub by anti-Myc beads and then incubated with Flag-USP39 or Flag-USP39 (C136/139A) mutant proteins immunoprecipitated by anti-Flag beads in vitro at 37˚C for 2 h with deubiquitination buffer, followed by immunoblotting as indicated. (E) 293T cells were transfected with vector, Flag-USP39, and Flag-USP39 (C136/C139A). After 48 h of transfection, cells were infected with VSV-GFP (MOI = 1.0) for 12 h. Viral level, IFN-b, IFIT1, and ISG54 mRNA levels were analyzed by real-time PCR. **p , 0.01, ***p , 0.001. A p value . 0.05 was considered ns.

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

Article Title: USP39 Serves as a Deubiquitinase to Stabilize STAT1 and Sustains Type I IFN-Induced Antiviral Immunity.

doi: 10.4049/jimmunol.1901384

Figure Lengend Snippet: FIGURE 5. USP39 stabilizes STAT1 protein level. (A) 293T cells were transfected with either vector or Flag-USP39 and then treated with CHX (50 mg/ml) for the indicated times. Cell lysates were analyzed by immunoblotting as indicated. (B) 293T cells were transfected with either control siRNA or siUSP39, together with HA-Ub plasmids. STAT1 proteins were immunoprecipitated, and STAT1 ubiquitination levels were analyzed. (C) 293T cells were cotransfected with Flag-STAT1 and HA-Ub or Flag-USP39 expression plasmids, respectively. Forty-eight hours posttransfection, the Flag-STAT1 and Flag- USP39 proteins were purified from 293T cells by anti-Flag beads. Both immunoprecipitated proteins were incubated at 37˚C for 2 h with deubiquitination buffer, followed by immunoblotting as indicated for in vitro deubiquitination assay. (D) Myc-STAT1 proteins were purified from 293T cells cotransfected with Myc-STAT1 and HA-Ub by anti-Myc beads and then incubated with Flag-USP39 or Flag-USP39 (C136/139A) mutant proteins immunoprecipitated by anti-Flag beads in vitro at 37˚C for 2 h with deubiquitination buffer, followed by immunoblotting as indicated. (E) 293T cells were transfected with vector, Flag-USP39, and Flag-USP39 (C136/C139A). After 48 h of transfection, cells were infected with VSV-GFP (MOI = 1.0) for 12 h. Viral level, IFN-b, IFIT1, and ISG54 mRNA levels were analyzed by real-time PCR. **p , 0.01, ***p , 0.001. A p value . 0.05 was considered ns.

Article Snippet: The following Abs were used: Abs against USP39 (1:2000; ab131244; Abcam, Cambridge, MA), IFNR1 (1:1000; DF6571; Affinity, Cincinnati, OH), IFNR2 (1:1000; sc-271105; Santa Cruz Biotechnology, Santa Cruz, CA), STAT1 (1:1000; catalog no. 9172; Cell Signaling Technology [CST], Danvers, MA), pY701-STAT1 (1:1000; catalog no. 9167; CST), STAT2 (1:1000; catalog no. 72704; CST), pY690-STAT2 (1:1000; catalog no. 88410; CST), JAK1 (1:1000; sc-1677; Santa Cruz Biotechnology), pJAK1 (1:1000; catalog no. 3331s; CST), Tyk2 (1:1000; catalog no. 14193; CST), p-Tyk2 (1:1000; catalog no. 9321; CST), VSV-G (1:5000; sc-66180; Santa Cruz Biotechnology), HA (1:2000; ab9110; Abcam), ubiquitin (1:1000; sc-8017; Santa Cruz Biotechnology), Flag (1:2500; F7425; Sigma-Aldrich), Myc (1:2000; catalog no. 9B11; CST), GAPDH (1:20,000; G9545; Sigma-Aldrich), tubulin (1:5000; 66031-1-Ig; Proteintech, Rosemont, IL).

Techniques: Transfection, Plasmid Preparation, Western Blot, Control, Immunoprecipitation, Ubiquitin Proteomics, Expressing, Incubation, In Vitro, Mutagenesis, Infection, Real-time Polymerase Chain Reaction

FIGURE 6. USP39 decreases K6-linked ubiquitination of STAT1. (A) 293T cells were transfected with Flag- STAT1, together with HA-Ub-K6, HA-Ub-K11, HA- Ub-K27, HA-Ub-K29, HA-Ub-K33, HA-Ub-K48 or HA-Ub-K63, and siUSP39. Immunoprecipitation was performed using anti-Flag M2 beads, followed by im- munoblotting using an anti-HA Ab. (B) 293T cells seeded in the 6-cm dish were transfected with HA vector or HA-K6, together with Flag-STAT1 plasmids. Thirty-six hours posttransfection, cells were treated with DMSO or MG132 (10 mM) for 6 and 12 h. Cell lysates were incubated with anti-Flag beads at 4˚C overnight, and the K6-linked ubiquitination of STAT1 was analyzed by Western blot. Data are representative of three independent experiments.

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

Article Title: USP39 Serves as a Deubiquitinase to Stabilize STAT1 and Sustains Type I IFN-Induced Antiviral Immunity.

doi: 10.4049/jimmunol.1901384

Figure Lengend Snippet: FIGURE 6. USP39 decreases K6-linked ubiquitination of STAT1. (A) 293T cells were transfected with Flag- STAT1, together with HA-Ub-K6, HA-Ub-K11, HA- Ub-K27, HA-Ub-K29, HA-Ub-K33, HA-Ub-K48 or HA-Ub-K63, and siUSP39. Immunoprecipitation was performed using anti-Flag M2 beads, followed by im- munoblotting using an anti-HA Ab. (B) 293T cells seeded in the 6-cm dish were transfected with HA vector or HA-K6, together with Flag-STAT1 plasmids. Thirty-six hours posttransfection, cells were treated with DMSO or MG132 (10 mM) for 6 and 12 h. Cell lysates were incubated with anti-Flag beads at 4˚C overnight, and the K6-linked ubiquitination of STAT1 was analyzed by Western blot. Data are representative of three independent experiments.

Article Snippet: The following Abs were used: Abs against USP39 (1:2000; ab131244; Abcam, Cambridge, MA), IFNR1 (1:1000; DF6571; Affinity, Cincinnati, OH), IFNR2 (1:1000; sc-271105; Santa Cruz Biotechnology, Santa Cruz, CA), STAT1 (1:1000; catalog no. 9172; Cell Signaling Technology [CST], Danvers, MA), pY701-STAT1 (1:1000; catalog no. 9167; CST), STAT2 (1:1000; catalog no. 72704; CST), pY690-STAT2 (1:1000; catalog no. 88410; CST), JAK1 (1:1000; sc-1677; Santa Cruz Biotechnology), pJAK1 (1:1000; catalog no. 3331s; CST), Tyk2 (1:1000; catalog no. 14193; CST), p-Tyk2 (1:1000; catalog no. 9321; CST), VSV-G (1:5000; sc-66180; Santa Cruz Biotechnology), HA (1:2000; ab9110; Abcam), ubiquitin (1:1000; sc-8017; Santa Cruz Biotechnology), Flag (1:2500; F7425; Sigma-Aldrich), Myc (1:2000; catalog no. 9B11; CST), GAPDH (1:20,000; G9545; Sigma-Aldrich), tubulin (1:5000; 66031-1-Ig; Proteintech, Rosemont, IL).

Techniques: Ubiquitin Proteomics, Transfection, Immunoprecipitation, Plasmid Preparation, Incubation, Western Blot

FIGURE 8. A proposed model of the regulation of STAT1 by USP39 through ubiquitin proteasome system. Without viral infection, the cytoplasmic STAT1 protein level was regulated by USP13 to reduce the STAT1 ubiquitination or by Smurf1 and SLIM to induce the ubiquitination of STAT1, which sustains the STAT1 protein level. USP39 is required for the stable association of the U4/U6·U5 trisnRNP to the prespliceosome, which is essential for pre- mRNA splicing. USP39 can promote tumor growth through the Wnt/b-catenin pathway or the AKT signaling pathway, etc. When the cells are infected with viruses, USP39 binds to nuclear STAT1 and cleaves K6-linked ubiquitin chains of STAT1 and therefore sustains STAT1 levels in the nucleus. STAT1 exports from the nucleus to the cytoplasm. IFNs promote the phosphorylation of STAT1 and the nuclear import of p-STAT1; ISGF3 binds to ISRE promoter to induce the expression of ISGs. Finally, these ISGs perform antiviral function.

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

Article Title: USP39 Serves as a Deubiquitinase to Stabilize STAT1 and Sustains Type I IFN-Induced Antiviral Immunity.

doi: 10.4049/jimmunol.1901384

Figure Lengend Snippet: FIGURE 8. A proposed model of the regulation of STAT1 by USP39 through ubiquitin proteasome system. Without viral infection, the cytoplasmic STAT1 protein level was regulated by USP13 to reduce the STAT1 ubiquitination or by Smurf1 and SLIM to induce the ubiquitination of STAT1, which sustains the STAT1 protein level. USP39 is required for the stable association of the U4/U6·U5 trisnRNP to the prespliceosome, which is essential for pre- mRNA splicing. USP39 can promote tumor growth through the Wnt/b-catenin pathway or the AKT signaling pathway, etc. When the cells are infected with viruses, USP39 binds to nuclear STAT1 and cleaves K6-linked ubiquitin chains of STAT1 and therefore sustains STAT1 levels in the nucleus. STAT1 exports from the nucleus to the cytoplasm. IFNs promote the phosphorylation of STAT1 and the nuclear import of p-STAT1; ISGF3 binds to ISRE promoter to induce the expression of ISGs. Finally, these ISGs perform antiviral function.

Article Snippet: The following Abs were used: Abs against USP39 (1:2000; ab131244; Abcam, Cambridge, MA), IFNR1 (1:1000; DF6571; Affinity, Cincinnati, OH), IFNR2 (1:1000; sc-271105; Santa Cruz Biotechnology, Santa Cruz, CA), STAT1 (1:1000; catalog no. 9172; Cell Signaling Technology [CST], Danvers, MA), pY701-STAT1 (1:1000; catalog no. 9167; CST), STAT2 (1:1000; catalog no. 72704; CST), pY690-STAT2 (1:1000; catalog no. 88410; CST), JAK1 (1:1000; sc-1677; Santa Cruz Biotechnology), pJAK1 (1:1000; catalog no. 3331s; CST), Tyk2 (1:1000; catalog no. 14193; CST), p-Tyk2 (1:1000; catalog no. 9321; CST), VSV-G (1:5000; sc-66180; Santa Cruz Biotechnology), HA (1:2000; ab9110; Abcam), ubiquitin (1:1000; sc-8017; Santa Cruz Biotechnology), Flag (1:2500; F7425; Sigma-Aldrich), Myc (1:2000; catalog no. 9B11; CST), GAPDH (1:20,000; G9545; Sigma-Aldrich), tubulin (1:5000; 66031-1-Ig; Proteintech, Rosemont, IL).

Techniques: Ubiquitin Proteomics, Infection, Phospho-proteomics, Expressing