stat1 rabbit polyclonal ab  (Cell Signaling Technology Inc)

Journal: The Journal of Biological Chemistry

Article Title: P300/RNA polymerase II mediates induction of the teleost viral RNA sensor MDA5 through the interferon regulatory factor IRF11

doi: 10.1016/j.jbc.2025.108193

Figure Lengend Snippet: Lc MDA5 is regulated by Lc IRF11. A and B , LYCK cells (5 × 10 6 ) were treated with r Lc IFNi (250 ng/ml) (A) or transfected with poly(I:C) (15 μg) (B) at different time points. The mRNA and protein levels of Lc MDA5 were examined using qRT-PCR and Western blot. Relative mRNA expression levels were normalized to β-actin and presented as fold induction compared to control cells set at 1. C , cells were pretreated with either DMSO or ruxolitinib (10 μM) for 30 min, followed by exposure to r Lc IFNi (250 ng/ml) for 12 h. Lc STAT1 bound with pan-phos was analyzed through Co-IP assay with anti-pan phospho-serine/threonine antibody. D , following the treatments described in ( C ), the expression of Lc MDA5 was measured using qPCR. E , cells were treated with ruxolitinib for 30 min, followed by transfection with 15 μg of poly(I:C) for 24 h to assess Lc MDA5 expression. F , LYCK cells (1 × 10 7 ), pre-treated with either DMSO or ruxolitinib, were electroporated with 10 μg of Flag-tagged Lc IRF11 for 36 h. Subsequently, cells were treated with r Lc IFNi for 12 h, followed by cell lysis for Phos-tag SDS-PAGE analysis. The phosphorylated Lc IRF11 level was normalized to the total Lc IRF11 level. Please note that in Phos-tag SDS-PAGE, molecular weight estimations using molecular weight markers are not feasible. G , cells electroporated with 10 μg of Flag-tagged Lc IRF11 or Lc IRF1 plasmid for 48 h were harvested and subjected to qPCR analysis to assess Lc MDA5 expression. H , cells were transfected with siRNAs (100 nM of each siRNA) against Lc IRF11 or Lc IRF1 or with negative control siRNA (siNC). Forty eight hours later, cells were harvested and subjected to qPCR analysis. I and J , cells were transfected with indicated siRNAs for 24 h, followed by treatment with r Lc IFNi (250 ng/ml) for 12 h ( I ). Similar experiments were carried out, only replacing r Lc IFNi with poly(I:C) (15 μg; by transfection) ( J ). Harvested cells were analyzed by qPCR. Error bars represent mean ± SD from three biological replicates. ∗ p < 0.05, two-tailed unpaired Student's t test.

Article Snippet: The Abs utilized in this study were as follows: Flag mouse monoclonal Ab (MBL, cat#M185-3L, 1:10,000 for Western blot); STAT1 rabbit polyclonal Ab (Cell Signaling Technology, cat#9172S, 1:1000 for Western blot); H3K4me3 rabbit polyclonal Ab (Thermo Fisher Scientific, cat#711958, 3 μg for ChIP); H3K27ac rabbit polyclonal Ab (Thermo Fisher Scientific, cat#PA5-96618, 3 μg for ChIP); p300 rabbit monoclonal Ab (Cell Signaling Technology, cat#57625, 1:100 for ChIP, 1:1000 for Western blot); POLR1A mouse monoclonal Ab (Pol II subunit RPB1, 8WG16, Thermo Fisher Scientific, cat#MA1-26249, 3 μg for ChIP, 1:1000 for Western blot); β-tubulin mouse monoclonal Ab (Beyotime; cat#AF2839, 1:1000 for Western blot), and HRP-conjugated goat anti mouse/rabbit secondary Abs (Proteintech, cat#SA00001-1, cat#SA00001-2, 1:10,000).

Techniques: Transfection, Quantitative RT-PCR, Western Blot, Expressing, Control, Co-Immunoprecipitation Assay, Lysis, SDS Page, Molecular Weight, Plasmid Preparation, Negative Control, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: P300/RNA polymerase II mediates induction of the teleost viral RNA sensor MDA5 through the interferon regulatory factor IRF11

doi: 10.1016/j.jbc.2025.108193

Figure Lengend Snippet: Lc IRF11 regulates Lc MDA5 promoter activity through binding to its promoter via its DBD domain. A , sequence alignments of the ISRE motif in the promoter region of fish MDA5. B , diagram of the Lc MDA5 promoter reporter plasmids showing WT and mutant type (mut) ISRE motifs. C , poly(I:C)-induced activation of Lc MDA5 promoter activity. EPC cells (1 × 10 6 ) in 24-well plates were transfected with the Lc MDA5 promoter reporter (250 ng) and pRL-TK (25 ng) for 6 h, followed by transfection with 1250 ng of poly(I:C) for 24 h to assess luciferase activity. D , Lc IRF11 induced Lc MDA5 promoter activity. EPC cells were cotransfected with pRL-TK, Lc MDA5 promoter reporter, and either Lc IRF11, Lc STAT1 expression plasmids, or control plasmid (pCMV-FLAG). Luciferase activity was measured at 24 h later. E , activation of Lc MDA5pro-luc and its ISRE mutant ( Lc MDA5pro-mut) by Lc IRF11. EPC cells were cotransfected with expression plasmids of pRL-TK, Lc IRF11, and either Lc MDA5pro-luc or its mutant for luciferase activity assay. F , activation of Lc MDA5 promoter by Lc IRF11-WT, but not Lc IRF11-ΔDBD. EPC cells were cotransfected with expression plasmids of pRL-TK, Lc MDA5 promoter reporter, Lc IRF11-WT, Lc IRF11-ΔDBD, or control plasmid. Luciferase activity was measured at 24 h post-transfection. G , Lc IRF11 bound to Lc MDA5 promoter DNA via its DBD domain. HEK293T cells (1 × 10 7 ) were transfected with 10 μg of indicated plasmids. After 24 h, cell lysates were mixed with 100 ng of biotinylated Lc MDA5 promoter DNA. Protein–DNA interactions were analyzed by Western blot. H , ChIP analysis confirmed the binding of Lc IRF11 to the Lc MDA5 promoter. HEK293T cells (1 × 10 7 ) were cotransfected with Lc MDA5 promoter reporter and Flag-tagged Lc IRF11-WT or Lc IRF11-ΔDBD (5 μg each) for 24 h. Following transfection, protein-bound DNA was immunoprecipitated using an anti-FLAG Ab. Subsequently, ChIP-enriched DNA was quantified by qPCR. I , Lc IRF11 binds to the ISRE motif of the Lc MDA5 promoter. HEK293T cells were transfected with the indicated plasmids. After incubating the biotinylated Lc MDA5 promoter DNA, the interaction of protein–DNA was detected by Western blot. Data represent the mean ± SD from three biological replicates. Asterisks indicate statistical significance (∗ p < 0.05, two-tailed unpaired Student's t test).

Article Snippet: The Abs utilized in this study were as follows: Flag mouse monoclonal Ab (MBL, cat#M185-3L, 1:10,000 for Western blot); STAT1 rabbit polyclonal Ab (Cell Signaling Technology, cat#9172S, 1:1000 for Western blot); H3K4me3 rabbit polyclonal Ab (Thermo Fisher Scientific, cat#711958, 3 μg for ChIP); H3K27ac rabbit polyclonal Ab (Thermo Fisher Scientific, cat#PA5-96618, 3 μg for ChIP); p300 rabbit monoclonal Ab (Cell Signaling Technology, cat#57625, 1:100 for ChIP, 1:1000 for Western blot); POLR1A mouse monoclonal Ab (Pol II subunit RPB1, 8WG16, Thermo Fisher Scientific, cat#MA1-26249, 3 μg for ChIP, 1:1000 for Western blot); β-tubulin mouse monoclonal Ab (Beyotime; cat#AF2839, 1:1000 for Western blot), and HRP-conjugated goat anti mouse/rabbit secondary Abs (Proteintech, cat#SA00001-1, cat#SA00001-2, 1:10,000).

Techniques: Activity Assay, Binding Assay, Sequencing, Mutagenesis, Activation Assay, Transfection, Luciferase, Expressing, Control, Plasmid Preparation, Western Blot, Immunoprecipitation, Two Tailed Test

Journal: Nature communications

Article Title: Heterozygous missense variant of the proteasome subunit β-type 9 causes neonatal-onset autoinflammation and immunodeficiency.

doi: 10.1038/s41467-021-27085-y

Figure Lengend Snippet: Fig. 1 Clinical figures and IFN signature of patients. a, b Pernio-like violaceous rash in patient 1 at 7-month-old (a) and patient 2 at 1-month-old (b). c–e myositis by leg magnetic resonance imaging (c), pulmonary hypertension by echocardiogram(d), and basal ganglia calcification by head computed tomography (CT) (e) in patient 1. f IFN scores of peripheral bloods of 11 healthy participants as controls, three Aicardi–Goutières syndrome (AGS) patients with IFIH1 variant and patient 2 at 84-day old. The score was regarded as positive if it exceeded +2 SD (5.04, dotted line) of the average (solid line) IFN score from healthy controls. Statistical analysis was performed with two-sided Mann–Whitney test. g The skin biopsy samples of a healthy participant as a control and patient 2 at 52-day-old stained with anti-p-STAT1 Ab. A scale bar, 100 μm. h The STAT1 phosphorylation assay with SV40-transformed dermal fibroblasts from healthy volunteers as controls and Patient 2. The expression levels of IFN-γ-induced p-STAT1 were enhanced in patient 2 fibroblasts compared to control fibroblasts.

Article Snippet: Expression of p-STAT1, STAT1, and β-actin was evaluated with anti-p-STAT1 (pY701) Ab (58D6, Cell Signaling 9167, 1:1000), rabbit polyclonal anti-STAT1 Abs (Stat1α (C-24), SANTA CRUZ sc-345, 1:1000) and anti-β-actin Ab (AC-74, Sigma-Aldrich A2228, AC-74, 1:2000), respectively.

Techniques: Magnetic Resonance Imaging, Computed Tomography, Variant Assay, MANN-WHITNEY, Control, Staining, Phospho-proteomics, Transformation Assay, Expressing

Journal: Nature communications

Article Title: Heterozygous missense variant of the proteasome subunit β-type 9 causes neonatal-onset autoinflammation and immunodeficiency.

doi: 10.1038/s41467-021-27085-y

Figure Lengend Snippet: Fig. 1 Clinical figures and IFN signature of patients. a, b Pernio-like violaceous rash in patient 1 at 7-month-old (a) and patient 2 at 1-month-old (b). c–e myositis by leg magnetic resonance imaging (c), pulmonary hypertension by echocardiogram(d), and basal ganglia calcification by head computed tomography (CT) (e) in patient 1. f IFN scores of peripheral bloods of 11 healthy participants as controls, three Aicardi–Goutières syndrome (AGS) patients with IFIH1 variant and patient 2 at 84-day old. The score was regarded as positive if it exceeded +2 SD (5.04, dotted line) of the average (solid line) IFN score from healthy controls. Statistical analysis was performed with two-sided Mann–Whitney test. g The skin biopsy samples of a healthy participant as a control and patient 2 at 52-day-old stained with anti-p-STAT1 Ab. A scale bar, 100 μm. h The STAT1 phosphorylation assay with SV40-transformed dermal fibroblasts from healthy volunteers as controls and Patient 2. The expression levels of IFN-γ-induced p-STAT1 were enhanced in patient 2 fibroblasts compared to control fibroblasts.

Article Snippet: Expression of p-STAT1, STAT1, and β-actin was evaluated with anti-p-STAT1 (pY701) Ab (58D6, Cell Signaling 9167, 1:1000), rabbit polyclonal anti-STAT1 Abs (Stat1α (C-24), SANTA CRUZ sc-345, 1:1000) and anti-β-actin Ab (AC-74, Sigma-Aldrich A2228, AC-74, 1:2000), respectively.

Techniques: Magnetic Resonance Imaging, Computed Tomography, Variant Assay, MANN-WHITNEY, Control, Staining, Phospho-proteomics, Transformation Assay, Expressing

Journal: Nature Communications

Article Title: Heterozygous missense variant of the proteasome subunit β-type 9 causes neonatal-onset autoinflammation and immunodeficiency

doi: 10.1038/s41467-021-27085-y

Figure Lengend Snippet: a, b Pernio-like violaceous rash in patient 1 at 7-month-old ( a ) and patient 2 at 1-month-old ( b ). c – e myositis by leg magnetic resonance imaging ( c ), pulmonary hypertension by echocardiogram( d ), and basal ganglia calcification by head computed tomography (CT) ( e ) in patient 1. f IFN scores of peripheral bloods of 11 healthy participants as controls, three Aicardi–Goutières syndrome (AGS) patients with IFIH1 variant and patient 2 at 84-day old. The score was regarded as positive if it exceeded +2 SD (5.04, dotted line) of the average (solid line) IFN score from healthy controls. Statistical analysis was performed with two-sided Mann–Whitney test. g The skin biopsy samples of a healthy participant as a control and patient 2 at 52-day-old stained with anti-p-STAT1 Ab. A scale bar, 100 μm. h The STAT1 phosphorylation assay with SV40-transformed dermal fibroblasts from healthy volunteers as controls and Patient 2. The expression levels of IFN-γ-induced p-STAT1 were enhanced in patient 2 fibroblasts compared to control fibroblasts.

Article Snippet: Expression of p-STAT1, STAT1, and β-actin was evaluated with anti-p-STAT1 (pY701) Ab (58D6, Cell Signaling 9167, 1:1000), rabbit polyclonal anti-STAT1 Abs (Stat1α (C-24), SANTA CRUZ sc-345, 1:1000) and anti-β-actin Ab (AC-74, Sigma-Aldrich A2228, AC-74, 1:2000), respectively.

Techniques: Magnetic Resonance Imaging, Computed Tomography, Variant Assay, MANN-WHITNEY, Control, Staining, Phospho-proteomics, Transformation Assay, Expressing