anti phospho stat1  (Santa Cruz Biotechnology)

Journal: Cell Communication and Signaling : CCS

Article Title: Characterization of a pathogenic gain-of-function mutation in the N-terminal domain of STAT1 which is reported to be associated with eosinophilic esophagitis

doi: 10.1186/s12964-025-02330-9

Figure Lengend Snippet: Localization of two N-terminal STAT1 gain-of-function mutations. (A) Position of two N-terminal GOF mutations D65 and D66 in the domain structure of STAT1 including the critical tyrosine residue Y701 in the transactivation domain (TAD). (B) Surface structure of the N-terminal dimer of STAT1 in orthogonal views and the ribbon diagram thereof. Shown is the localization of crucial residues involved in N-terminal interactions: F77 (magenta), D92 and E96 (both yellow) and the amino-acid residues under investigation D65 (light green) and D66 (dark green). The two monomers are shown in different colors. (C) Ribbon diagram of the crystal structure of the antiparallel tetramer including the two aspartic acid residues. The four N-terminal domains in the interior complex as well as the truncated core proteins are colored differently. Protein crystal structure renderings from the Protein Data Bank file 1YVL were generated using the PyMOL software (DeLano Scientific)

Article Snippet: The following primary antibodies were used, all diluted in 1:1000 in blocking solution (4% BSA in TBS-T): rabbit monoclonal antibodies against phospho-Tyr701-STAT1 (Cell Signaling Technology, 58D6), pan-STAT1 (Cell Signaling Technology, D1K9Y), and the mouse monoclonal anti-Flag antibody (Sigma Aldrich, M2).

Techniques: Residue, Generated, Software

Journal: Cell Communication and Signaling : CCS

Article Title: Characterization of a pathogenic gain-of-function mutation in the N-terminal domain of STAT1 which is reported to be associated with eosinophilic esophagitis

doi: 10.1186/s12964-025-02330-9

Figure Lengend Snippet: Hyperphosphorylation of the STAT1-D65A and -D66A mutants. Western blot results from whole-cell extracts using U3A cells (A–D , G , H) expressing GFP-tagged (A , B) , untagged (C , D) or Flag-tagged (G , H) STAT1 proteins and HeLa cells expressing STAT1-GFP (E , F) . Cells expressing either WT or mutant STAT1- D65A and -D66A were stimulated with 50 ng/ml of IFNγ for 45 min and then treated with 1 µM of the kinase inhibitor staurosporine for the indicated times. Immunoblots were probed with a phosphotyrosine-specific STAT1 antibody (α-pSTAT1) to assess STAT1 phosphorylation and a pan-STAT1 (α-STAT1) or Flag antibody (α-Flag) to detect total STAT1 protein levels. The quantifications of the blots from at least three independent experiments are shown in (B , D , F , H) . Statistically significant differences between WT protein and mutant STAT1 are indicated by asterisks

Article Snippet: The following primary antibodies were used, all diluted in 1:1000 in blocking solution (4% BSA in TBS-T): rabbit monoclonal antibodies against phospho-Tyr701-STAT1 (Cell Signaling Technology, 58D6), pan-STAT1 (Cell Signaling Technology, D1K9Y), and the mouse monoclonal anti-Flag antibody (Sigma Aldrich, M2).

Techniques: Western Blot, Expressing, Mutagenesis, Phospho-proteomics

Journal: Cell Communication and Signaling : CCS

Article Title: Characterization of a pathogenic gain-of-function mutation in the N-terminal domain of STAT1 which is reported to be associated with eosinophilic esophagitis

doi: 10.1186/s12964-025-02330-9

Figure Lengend Snippet: Increased gene-specific transcriptional activity of D65A and D66A. (A–C) STAT1-negative U3A cells were transfected with expression plasmids coding for GFP-tagged fusion proteins of WT, D65A or D66A variants in combination with a luciferase reporter gene construct (3xLy6E, pIC-339, or pIC-1352) and a constitutively expressed β-galactosidase plasmid used for normalization. Cells were stimulated with 50 ng/ml of IFNγ for 0 h (-) or 6 h (+) before extracts were tested for luciferase and β-galactosidase production. Significant differences between the WT and mutant proteins are marked with asterisks. (D–I) Real-time PCR analysis was performed to compare mRNA production of endogenous STAT1 target genes for WT and the tested mutants. Cells were treated with 50 ng/ml of IFNγ for 6 h (+) or left untreated (-). The following gene transcripts were examined: STAT1 (D) , IRF1 (E) , MCP1 (F) , MIG (G) , GBP1 (H) , and CXCL10 (I) . Significant differences between WT and the mutants are highlighted by asterisks

Article Snippet: The following primary antibodies were used, all diluted in 1:1000 in blocking solution (4% BSA in TBS-T): rabbit monoclonal antibodies against phospho-Tyr701-STAT1 (Cell Signaling Technology, 58D6), pan-STAT1 (Cell Signaling Technology, D1K9Y), and the mouse monoclonal anti-Flag antibody (Sigma Aldrich, M2).

Techniques: Activity Assay, Transfection, Expressing, Luciferase, Construct, Plasmid Preparation, Mutagenesis, Real-time Polymerase Chain Reaction

Journal: Cell Communication and Signaling : CCS

Article Title: Characterization of a pathogenic gain-of-function mutation in the N-terminal domain of STAT1 which is reported to be associated with eosinophilic esophagitis

doi: 10.1186/s12964-025-02330-9

Figure Lengend Snippet: Normal DNA-binding affinity of two N-terminal GOF mutants. (A–D) Kinetics of DNA binding to a [ 33 P]-labeled, single GAS site (M67) using extracts from reconstituted U3A cells (A , B) or HeLa cells (C , D) expressing either WT, D65A or D66A. Cells were stimulated with 50 ng/ml IFNγ for 45 min and subsequently treated with 1 µM staurosporine for the indicated times. Extracts were mixed with the M67 probe for 5 min before being separated by electrophoresis. Representative autoradiograms (A , C) including the quantification of three experiments (B , D) are shown. Note that the increased DNA-binding intensity of the mutants compared to WT reflects their elevated level of tyrosine phosphorylation, as demonstrated from the Western blot data in Fig. . Arrowheads indicate binding on DNA as dimers, and unspecific bands are marked with an asterisk. Asterisks in all quantification graphs represent significant differences observed between the WT protein and its respective mutants. (E) Representative autoradiogram from a competition gelshift experiment using U3A whole cell extracts expressing the indicated STAT1 variants and a 750-fold excess of unlabeled M67 as competitor. To ensure equal loading of phospho-proteins, extracts from mutant-expressing cells were diluted 1:2 with extracts from non-transfected U3A cells. The [ 33 P]-labeled M67 probe was incubated with the samples for 30 min, followed by exposure to the unlabeled M67 for increasing time intervals (0 min, 5 min, and 10 min) on ice. Arrowhead indicates binding on DNA as dimers, and an unspecific band is marked with an asterisk. (F) Quantification of EMSA competition results from three experiments as shown in (E) . Note the similar dissociation kinetics of the WT protein and the mutants. (G–I) Similar sequence-specific DNA binding of D65A and D66A compared to the WT molecule. Shown is a representative autoradiogram of a gelshift experiment using STAT1-reconstituted cells and different DNA probes with increasing affinity containing no GAS site (2xnon-GAS), one single GAS site (GAS-nonGAS) or two GAS sites in tandem orientation (2xGAS). Transfected cells expressing the indicated STAT1 variants were stimulated for 45 min with IFNγ, before the corresponding extracts were incubated for 5 min at RT with the [ 33 P]-labeled DNA probes. The different bands for tetramers and dimers are highlighted and an unspecific band is marked with an asterisk. (H , I) Quantification of normalized DNA-binding affinity for the three different tandem GAS elements on the tetrameric (H) and dimeric (I) position

Article Snippet: The following primary antibodies were used, all diluted in 1:1000 in blocking solution (4% BSA in TBS-T): rabbit monoclonal antibodies against phospho-Tyr701-STAT1 (Cell Signaling Technology, 58D6), pan-STAT1 (Cell Signaling Technology, D1K9Y), and the mouse monoclonal anti-Flag antibody (Sigma Aldrich, M2).

Techniques: Binding Assay, Labeling, Expressing, Electrophoresis, Phospho-proteomics, Western Blot, Mutagenesis, Transfection, Incubation, Sequencing

Journal: Cell Communication and Signaling : CCS

Article Title: Characterization of a pathogenic gain-of-function mutation in the N-terminal domain of STAT1 which is reported to be associated with eosinophilic esophagitis

doi: 10.1186/s12964-025-02330-9

Figure Lengend Snippet: STAT1-D65A and -D66A show prolonged nuclear accumulation. (A–D) HeLa cells expressing GFP-tagged STAT1-WT (A) or the respective mutant fusion proteins (B , C) were either left untreated or exposed to 50 ng/ml of IFNγ for 45 min followed by incubation with 1 µM staurosporine for the specified times (0 min, 30 min, 60 min). Cells were immunocytochemically stained using a phosphotyrosine-specific anti-STAT1 antibody. (D) Quantification of immunofluorescence intensities from at least 15 cells displayed as the ratio of nuclear-to-total phosphorylated STAT1. Significant differences between the WT protein and the mutants are marked by asterisks. (E–H) Similar experiments to those described in (A–D) except that U3A cells expressing STAT1-GFP-WT (E) or the respective mutants -D65A (F) and -D66A (G) were used. Note that in both cell types the two aspartic acid-to-alanine mutants show a more pronounced and prolonged phase of nuclear accumulation following cytokine exposure

Article Snippet: The following primary antibodies were used, all diluted in 1:1000 in blocking solution (4% BSA in TBS-T): rabbit monoclonal antibodies against phospho-Tyr701-STAT1 (Cell Signaling Technology, 58D6), pan-STAT1 (Cell Signaling Technology, D1K9Y), and the mouse monoclonal anti-Flag antibody (Sigma Aldrich, M2).

Techniques: Expressing, Mutagenesis, Incubation, Staining, Immunofluorescence

Journal: Arthritis Research & Therapy

Article Title: Altered phenotype and Stat1 expression in Toll-like receptor 7/8 stimulated monocyte-derived dendritic cells from patients with primary Sjögren’s syndrome

doi: 10.1186/ar4682

Figure Lengend Snippet: Stat1 expression is significantly reduced in TLR7/8 stimulated monocyte-derived dendritic cells from primary Sjögren’s syndrome patients. (A) , (B) Representative western blots for Stat1α, p-Stat1α (Y701), p-Stat1α (S727) and glyceraldehyde 3-phosphatase dehydrogenase (GAPDH) expression in mature monocyte-derived dendritic cells (moDC) from primary Sjögren’s syndrome (pSS) patients ( n = 4) and controls ( n = 4). (C) Stat1 expression in immature and mature moDC from patients ( n = 8) and controls ( n = 8). (D) p-Stat1α (S727) expression in immature and mature moDC from patients ( n = 8) and controls ( n = 8). (E) p-Stat1α (Y701) expression in immature and mature moDC from patients (P immature n = 7 and P mature n = 8) and controls (C immature n = 5 and C mature n = 8). Relative expression levels are presented as arbitrary units and were calculated as the percentage of band density normalized to GAPDH expression in relation to a standard sample used on all blots (set to 100%).

Article Snippet: Membranes were probed with antibodies against Ro52/SSA (sc-25351), IRF-8 (sc-6058), Bim (sc-11425), Stat1 (sc-464), p-Stat1 Tyr701 (sc-7988), p-Stat1 Ser727 (sc-16570), Stat3 (sc-482), pStat3 (sc-8059; all antibodies purchased from Santa Cruz Biotechnology, Santa Cruz, CA, USA), and GAPDH (clone 6C5; HyTest, Turku, Finland).

Techniques: Expressing, Derivative Assay, Western Blot