Journal: The Journal of general virology

Article Title: Epstein-Barr virus induces a distinct form of DNA-bound STAT1 compared with that found in interferon-stimulated B lymphocytes.

doi: 10.1099/vir.0.82741-0

Figure Lengend Snippet: Fig. 1. STAT1 is tyrosine-phosphorylated in LCLs after IFN-a stimulation, but can bind DNA in the absence of stimulation. (a) Total cell lysates were generated from three cell lines: Kit 225, KEM LCL and EB LCL. These cell lines were either unstimulated or incubated with IFN-a (1000 IU) for 30 min. These lysates were analysed by SDS-PAGE and Western blotting using antibodies specific to phospho-STAT1 (Y701), pan-STAT1 and LMP1. The Kit 225 T-cell line was used as a positive control for the presence of tyrosine- phosphorylated STAT1 following IFN-a stimulation, and LMP1 detection was used as a positive marker for EBV. (b) STAT1 DNA binding was measured in the BL41, BL41+B95.8 and IARC-171 cell lines by using a DNA-affinity precipitation assay. These cell lines were either unstimulated or incubated with IFN-a (1000 IU) for 30 min. DNA-bound proteins were analysed by SDS-PAGE and Western blotting using antibodies specific to phospho-STAT1 (Y701) and pan-STAT1. Typically, 1107 cell equivalents were applied to each lane of the gel. These results are representative of four separate experiments. (c) STAT1 DNA binding was measured in the BL41, BL41+B95.8 and IARC-171 cell lines by using an EMSA. These cell lines were either unstimulated or incubated with IFN-a (1000 IU) for 30 min. Nuclear extract (10 mg) was then incubated with 2 ng 32P-radiolabelled GRR oligonucleotide or SIE oligonucleotide probe. Protein–DNA complexes were separated by using a native 4 % polyacrylamide gel and visualized by autoradio- graphy. Only protein–DNA complexes are shown, as free probe has been removed from the figure. The arrow indicates a specific protein–DNA complex. The results shown are representative of three separate experiments.

Article Snippet: Antibodies to phospho-STAT1 (Y701) (sc-7988-R), phospho-STAT1 (S727) (sc-16570-R), pan-STAT1 (sc-346) and phospho-ERK1/2 (Y204) (sc-7383) ERK1/2 (sc-93) were from Santa Cruz Biotechnology and were used at a concentration of 0.2 mg ml21.

Techniques: Generated, Incubation, SDS Page, Western Blot, Positive Control, Marker, Binding Assay, Affinity Precipitation

Journal: The Journal of general virology

Article Title: Epstein-Barr virus induces a distinct form of DNA-bound STAT1 compared with that found in interferon-stimulated B lymphocytes.

doi: 10.1099/vir.0.82741-0

Figure Lengend Snippet: Fig. 2. EBV induces a constitutive STAT1 DNA-binding complex that is capable of stimulating transcriptional activation without requiring tyrosine phosphorylation. (a) Supershift analysis of protein–DNA complexes was measured in unstimulated and IFN-a- treated IARC-171 LCL cells. Nuclear protein (10 mg) was pre-incubated for 30 min with 2 mg STAT1 supershift antibody (sc- 592 X) prior to incubation with 2 ng 32P-radiolabelled GRR oligonucleotide or SIE oligonucleotide probe. Protein–DNA complexes were separated by using a native 4 % polyacrylamide gel and visualized by autoradiography. The results shown are representative of two separate experiments. (b) The specificity of STAT1–DNA complexes was measured in unstimulated and IFN-a-treated IARC-171 LCL cells. Nuclear protein (10 mg) was pre-incubated for 30 min with 2 mg STAT1 supershift antibody (sc-592 X) or 100 ng cold GRR oligonucleotide prior to incubation with 2 ng 32P-radiolabelled GRR oligonucleotide or mGRR oligonucleotide probe. Protein–DNA complexes were then separated by using a native 4 % polyacrylamide gel and were visualized by autoradiography. (c) Supershift of protein–DNA complexes was measured in unstimulated and IFN-a-treated IARC-171 LCL cells. Nuclear protein (10 mg) was pre-incubated for 30 min with 2 mg STAT1 supershift antibody (BD #610119) prior to incubation with 2 ng 32P-radiolabelled GRR oligonucleotide. Protein–DNA complexes were separated by using a native 4 % polyacrylamide gel and visualized by autoradiography. Arrows indicate specific protein–DNA and supershifted protein–DNA complexes. The results shown are representative of two separate experiments. (d) STAT transcriptional activation was measured in IARC-171 LCL cells by using a STAT reporter assay. Cells (1107) were transfected with 20 mg empty vector-luc reporter, 5 mg GRR (5)-luc reporter, 10 mg GRR (5)-luc reporter or 20 mg GRR (5)-luc reporter. One microgram of phRL-SV40 reporter was also co-transfected and luciferase activity was assayed 20 h post-transfection. Relative luciferase activity was calculated as a ratio of firefly over Renilla luciferase. The results are mean values representative of at least three experiments. Error bars indicate 1 SEM.

Article Snippet: Antibodies to phospho-STAT1 (Y701) (sc-7988-R), phospho-STAT1 (S727) (sc-16570-R), pan-STAT1 (sc-346) and phospho-ERK1/2 (Y204) (sc-7383) ERK1/2 (sc-93) were from Santa Cruz Biotechnology and were used at a concentration of 0.2 mg ml21.

Techniques: Binding Assay, Activation Assay, Phospho-proteomics, Incubation, Autoradiography, Reporter Assay, Transfection, Plasmid Preparation, Luciferase, Activity Assay

Journal: The Journal of general virology

Article Title: Epstein-Barr virus induces a distinct form of DNA-bound STAT1 compared with that found in interferon-stimulated B lymphocytes.

doi: 10.1099/vir.0.82741-0

Figure Lengend Snippet: Fig. 3. LMP1 induces STAT1 protein expression, nuclear translo- cation and DNA binding without triggering tyrosine phosphoryla- tion. (a) LMP1 expression was measured in stable DG75 transfectants (with inducible LMP1 expression) following removal of 1 mg tetracycline ml”1. Total lysates were generated from cells that were washed five times in RPMI 1640 medium and recultured in the presence of tetracycline (+) or absence of tetracycline for either 72 h (”3) or 96 h (”4). IARC-171 LCLs were used as a positive control for LMP1. These lysates were analysed by SDS- PAGE and Western blotting using antibodies specific to LMP1 and actin. Typically, 2105 cells were applied to each lane of the gel. These results are representative of three experiments. (b) STAT1 tyrosine phosphorylation and nuclear expression were measured in stable DG75 transfectants with inducible LMP1 expression. These cells were recultured in the presence of tetracycline (+) or absence of tetracycline for either 72 h (”3) or 96 h (”4). Cells were also incubated with IFN-a (1000 IU) for 30 min or left unstimulated. STAT1 tyrosine phosphorylation and nuclear expression were also measured in unstimulated IARC-171 LCL cells. Nuclear extracts were generated and were then analysed by SDS-PAGE and Western blotting using antibodies specific to phospho-STAT1 (Y701), pan-STAT1 and actin. Nuclear extract (10 mg) was applied to each lane of the gel. These results are representative of three experiments. (c) STAT1 DNA binding was measured in stable DG75 transfectants with inducible LMP1 expression by using an EMSA. These cells were recultured in the presence of tetracycline (+) or absence of tetracycline for either 72 h (”3) or 96 h (”4). STAT1 DNA binding was also measured in IARC-171 LCL cells. Cells were incubated with IFN-a for 30 min or left unstimulated. Nuclear extract (10 mg) was incubated with 2 ng 32P-radiolabelled GRR oligonucleotide probe. Protein–DNA complexes were separated by using a native 4 % polyacrylamide gel and visualized by autoradiography. Only the protein–DNA complexes are shown, as free probe has been removed from the figure. The arrow indicates a specific protein– DNA complex. The results shown are representative of three separate experiments.

Article Snippet: Antibodies to phospho-STAT1 (Y701) (sc-7988-R), phospho-STAT1 (S727) (sc-16570-R), pan-STAT1 (sc-346) and phospho-ERK1/2 (Y204) (sc-7383) ERK1/2 (sc-93) were from Santa Cruz Biotechnology and were used at a concentration of 0.2 mg ml21.

Techniques: Expressing, Binding Assay, Generated, Positive Control, SDS Page, Western Blot, Phospho-proteomics, Incubation, Autoradiography

Journal: The Journal of general virology

Article Title: Epstein-Barr virus induces a distinct form of DNA-bound STAT1 compared with that found in interferon-stimulated B lymphocytes.

doi: 10.1099/vir.0.82741-0

Figure Lengend Snippet: Fig. 4. STAT1 is constitutively serine-phosphorylated in LCLs, but lacks detectable lysine acetylation. (a) STAT1 immunoprecipitates were generated from two B-cell lines, DG75 and IARC-171 LCL. These cell lines were either unstimulated or incubated with IFN-a (1000 IU) for 30 min. Beads only and irrelevant antibody (Irr. Ab; ATF-3) controls were also incubated with nuclear extracts of untreated IARC-171 LCL cells. STAT1 immunoprecipitates were then analysed by SDS-PAGE and Western blotting using antibodies specific to phospho-STAT1 (S727) and pan-STAT1. Typically, 5106 cell equivalents were loaded in each lane of the gel. These results are representative of three separate experiments. (b) The presence of serine- phosphorylated STAT1 in DNA-bound protein complexes was measured in unstimulated and IFN-a-treated IARC-171 LCL cells. Nuclear protein (10 mg) was pre-incubated for 30 min with 2 mg phospho-STAT1 (S727) antibody prior to incubation with 2 ng 32P-radiolabelled GRR oligonucleotide. Protein–DNA complexes were separated by using a native 4 % polyacrylamide gel and visualized by autoradiography. Arrows indicate specific protein–DNA and supershifted protein–DNA complexes. The results shown are representative of two separate experiments. (c) STAT1 immunoprecipitates were generated from IARC-171 LCL cells that were unstimulated, incubated with IFN-a (1000 IU) for 30 min and/or treated with trichostatin A (TSA) (2 mM) for 24 h. STAT1 immunoprecipitates were then analysed by SDS-PAGE and Western blotting using antibodies specific to acetyl- lysine and pan-STAT1. Typically, 5106 cell equivalents were loaded in lanes 1–6 of the gel, and 2.5105 cell equivalents of nuclear extracts from unstimulated and TSA-treated IARC-171 LCL cells were loaded in lanes 7 and 8 as controls. These results are representative of four separate experiments.

Article Snippet: Antibodies to phospho-STAT1 (Y701) (sc-7988-R), phospho-STAT1 (S727) (sc-16570-R), pan-STAT1 (sc-346) and phospho-ERK1/2 (Y204) (sc-7383) ERK1/2 (sc-93) were from Santa Cruz Biotechnology and were used at a concentration of 0.2 mg ml21.

Techniques: Generated, Incubation, SDS Page, Western Blot, Autoradiography

Journal: The Journal of general virology

Article Title: Epstein-Barr virus induces a distinct form of DNA-bound STAT1 compared with that found in interferon-stimulated B lymphocytes.

doi: 10.1099/vir.0.82741-0

Figure Lengend Snippet: Fig. 5. STAT1 is serine-phosphorylated downstream of PI3K and MEK and seems to restrict IFN-stimulated STAT1 DNA binding. (a) Total cell lysates were generated from IARC-171 LCL cells incubated for 24 h with different combinations of PD98059 and LY294002. These combinations were: PD98059 (50 mM) alone; LY294002 (20 mM) alone; and PD98059 (50 mM)+LY294002 (20 mM). Total cell lysates were incubated with DMSO for 24 h as a control. These lysates were then analysed by SDS-PAGE and Western blotting using antibodies specific to phospho-STAT1 (S727), pan-STAT1, phospho- ERK1/2 (Y204), pan-ERK1/2, phospho-S6, pan-S6 and actin. Typically, 5105 cells were applied to each lane of the gel. These results are representative of four experiments. (b) The effect of serine phosphorylation on STAT1 DNA binding was measured in IARC-171 LCL cells by using an EMSA. These cells were unstimulated, treated with a combination of PD98059 (50 mM) and LY294002 (20 mM) for 24 h and/or incubated with IFN-a (1000 IU) for 30 min. Nuclear extract (10 mg) was then incubated with 2 ng 32P-radiolabelled GRR oligonucleotide probe. Protein–DNA complexes were separated by using a native 4 % polyacrylamide gel and visualized by autoradiography. Nuclear extract (10 mg) was also analysed by SDS-PAGE and Western blotting using antibodies specific to STAT1 and actin. This demonstrates that the nuclear levels of STAT1 were equal in each sample analysed. The results shown are representative of five separate experiments.

Article Snippet: Antibodies to phospho-STAT1 (Y701) (sc-7988-R), phospho-STAT1 (S727) (sc-16570-R), pan-STAT1 (sc-346) and phospho-ERK1/2 (Y204) (sc-7383) ERK1/2 (sc-93) were from Santa Cruz Biotechnology and were used at a concentration of 0.2 mg ml21.

Techniques: Binding Assay, Generated, Incubation, Control, SDS Page, Western Blot, Phospho-proteomics, Autoradiography

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