pan stat1  (Cell Signaling Technology Inc)

Journal: Cell communication and signaling : CCS

Article Title: A novel interface between the N-terminal and coiled-coil domain of STAT1 functions in an auto-inhibitory manner.

doi: 10.1186/s12964-023-01124-1

Figure Lengend Snippet: Fig. 2 Tyrosine-phosphorylated STAT1-E169A shows prolonged nuclear accumulation. HeLa cells expressing fusion proteins of STAT1-WT (A) or the H158A (B) and E169A (C) mutants were either left unstimulated or stimulated with 50 ng/ml IFNγ for 45 min, this being followed by incubation with staurosporine (1 µM) for the indicated times (scale bar corresponds to 50 µm). D Quantification of the results from Fig. 2A–C. STAT1-GFP fluorescence intensity data from n = 20 cells are shown as the ratio of nuclear-to-total STAT1. Asterisks indicate significant differences between the WT protein and the respective mutant

Article Snippet: The membranes were blocked with 4% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) for 1 h and then incubated overnight at 4 °C with rabbit monoclonal antibodies directed against either phospho-Tyr701-STAT1 (Cell Signaling Technology, 58D6) or pan-STAT1 (Cell Signaling Technology, D1K9Y), both diluted 1:1000 in blocking solution.

Techniques: Expressing, Incubation, Fluorescence, Mutagenesis

Journal: Cell communication and signaling : CCS

Article Title: A novel interface between the N-terminal and coiled-coil domain of STAT1 functions in an auto-inhibitory manner.

doi: 10.1186/s12964-023-01124-1

Figure Lengend Snippet: Fig. 3 STAT1-E169A retains high levels of tyrosine phosphorylation and nuclear localization. U3A cells expressing fusion proteins of WT (A) or mutant (B, C) STAT1 were either not stimulated or stimulated with 50 ng/ml IFNγ for 45 min before incubation with 1 µM staurosporine for the indicated times (scale bar corresponds to 50 µm). D Quantification of the results from Fig. 3A–C. Total fluorescence intensity of tyrosine-phosphorylated STAT1 was measured from n = 20 cells in each experiment. Asterisks indicate significant differences in the tyrosine-phosphorylation level between the WT protein and the E169A mutant

Article Snippet: The membranes were blocked with 4% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) for 1 h and then incubated overnight at 4 °C with rabbit monoclonal antibodies directed against either phospho-Tyr701-STAT1 (Cell Signaling Technology, 58D6) or pan-STAT1 (Cell Signaling Technology, D1K9Y), both diluted 1:1000 in blocking solution.

Techniques: Phospho-proteomics, Expressing, Mutagenesis, Incubation, Fluorescence

Journal: Cell communication and signaling : CCS

Article Title: A novel interface between the N-terminal and coiled-coil domain of STAT1 functions in an auto-inhibitory manner.

doi: 10.1186/s12964-023-01124-1

Figure Lengend Snippet: Fig. 4 Nuclear accumulation of the STAT1-E169A mutant is accelerated after IFNγ stimulation. HeLa cells expressing fusion proteins of STAT1-WT (A) or the H158A (B) and E169A (C) mutants were either left unstimulated or stimulated with 50 ng/ml IFNγ for the indicated times (scale bar corresponds to 50 µm). D Quantification of the results from Fig. 4A–C. STAT1-GFP fluorescence intensity data from n = 20 cells are plotted as the ratio of nuclear-to-total STAT1. Asterisks indicate significant differences between the WT protein and the respective mutant

Article Snippet: The membranes were blocked with 4% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) for 1 h and then incubated overnight at 4 °C with rabbit monoclonal antibodies directed against either phospho-Tyr701-STAT1 (Cell Signaling Technology, 58D6) or pan-STAT1 (Cell Signaling Technology, D1K9Y), both diluted 1:1000 in blocking solution.

Techniques: Mutagenesis, Expressing, Fluorescence

Journal: Cell communication and signaling : CCS

Article Title: A novel interface between the N-terminal and coiled-coil domain of STAT1 functions in an auto-inhibitory manner.

doi: 10.1186/s12964-023-01124-1

Figure Lengend Snippet: Fig. 5 The STAT1-E169A mutation increases transcriptional activity of target genes. A U3A cells coexpressing fusion proteins of WT or mutant STAT1 with a luciferase reporter gene coupled to a 3xLy6E, pIC-339 or pIC-1352 promoter were stimulated for 6 h with 50 ng/ml IFNγ. The data are normalized to β-galactosidase coexpression. B mRNA levels of endogenous STAT1 target genes in IFNγ-stimulated U3A cells expressing WT or mutant STAT1, obtained by qPCR. Asterisks indicate significant differences between the WT protein and the respective mutant

Article Snippet: The membranes were blocked with 4% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) for 1 h and then incubated overnight at 4 °C with rabbit monoclonal antibodies directed against either phospho-Tyr701-STAT1 (Cell Signaling Technology, 58D6) or pan-STAT1 (Cell Signaling Technology, D1K9Y), both diluted 1:1000 in blocking solution.

Techniques: Mutagenesis, Activity Assay, Luciferase, Expressing

Journal: Cell communication and signaling : CCS

Article Title: A novel interface between the N-terminal and coiled-coil domain of STAT1 functions in an auto-inhibitory manner.

doi: 10.1186/s12964-023-01124-1

Figure Lengend Snippet: Fig. 7 A novel mechanism for the dissociation of STAT1 from DNA through auto-inhibition. Surface representation of a tyrosine-phosphorylated parallel STAT1 dimer (yellow + green) binding to DNA (orange). The position of the respective N-terminal domains (N) is approximated. The critical amino acid position E169 (magenta) in the CCD is marked with arrows. According to the model, binding of the N-terminus to the E169 residue of its own core fragment facilitates dissociation from DNA

Article Snippet: The membranes were blocked with 4% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) for 1 h and then incubated overnight at 4 °C with rabbit monoclonal antibodies directed against either phospho-Tyr701-STAT1 (Cell Signaling Technology, 58D6) or pan-STAT1 (Cell Signaling Technology, D1K9Y), both diluted 1:1000 in blocking solution.

Techniques: Inhibition, Binding Assay, Residue

Journal: Frontiers in Pharmacology

Article Title: Influenza A Virus-Driven Airway Inflammation may be Dissociated From Limb Muscle Atrophy in Cigarette Smoke-Exposed Mice

doi: 10.3389/fphar.2022.859146

Figure Lengend Snippet: Effect of CSE and poly I:C on inflammatory response of lung epithelial cells. BEAS2B cells were exposed to polyinosinic:polycytidylic acid (poly I:C), 25% cigarette smoke extract (CSE), or a combination of the two for 1, 2 and 6 h. (A) Western blot images of phospo-STAT1, pan-STAT1, phospho-JNK and pan-JNK. (B) qPCR analysis was performed to assess expression of inflammatory markers Ifnβ, Il6 and Tnfα. Data shown as mean +SEM ( n = 7–9 per group). **** p < 0.0001.

Article Snippet: Western blot analyses were performed on 12% SDS PAGE gels to assess key signalling molecules including phospho-/pan- STAT1, phospho-/pan- JNK, GAPDH, BiP, phospho-/pan-eIF2α, phospho-/pan-p70S6 kinase, phospho-/pan- S6rp, phospho-/pan- 4E-BP1, tubulin (Cell Signaling Technology, United States), MAFbx and MURF-1 (Abcam, United States).

Techniques: Western Blot, Expressing

Journal: Journal of Virology

Article Title: Serial Passaging of the Human Rotavirus CDC-9 Strain in Cell Culture Leads to Attenuation: Characterization from In Vitro and In Vivo Studies

doi: 10.1128/JVI.00889-20

Figure Lengend Snippet: Wild-type CDC-9 induces increased STAT1 phosphorylation in Caco-2 cells. Caco-2 cells were infected with CDC-9 P11, P28, and P44 at an MOI of 0.2, harvested at 6 and 24 hpi, and analyzed for STAT1 phosphorylation (p-STAT1) by enzyme immunoassay (A) and Western blotting (B) as described in the text. The data in panel A for p-STAT1 are the averages and 3 standard errors from 2 experiments (n = 9) compared to pan-STAT1 expression. Images in panel B were generated from three different Western blots. Mock or RRV infections were used for controls. Statistical analysis was done to compare levels of STAT1 phosphorylation to that of CDC-9 infection and the mock infection control using a paired t test. ns, not significant (P > 0.05); *, P ≤ 0.05; ***, P < 0.001.

Article Snippet: The phosphorylation of the transcription factor STAT1 was analyzed by using the phosphor-STAT1 (pSer 727 ) and pan-STAT1 enzyme-linked immunosorbent assay (ELISA) kit (Sigma-Aldrich, St. Louis, MO) according to the manufacturer’s instructions.

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Generated