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    Santa Cruz Biotechnology pan stat1 sc 346
    Fig. 1. <t>STAT1</t> 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.
    Pan Stat1 Sc 346, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 96/100, based on 2371 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Epstein-Barr virus induces a distinct form of DNA-bound STAT1 compared with that found in interferon-stimulated B lymphocytes."

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

    Journal: The Journal of general virology

    doi: 10.1099/vir.0.82741-0

    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.
    Figure Legend 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.

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

    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.
    Figure Legend 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.

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

    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.
    Figure Legend 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.

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

    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.
    Figure Legend 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.

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

    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.
    Figure Legend 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.

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



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    pan stat1 sc 346  (Santa Cruz Biotechnology)
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    Santa Cruz Biotechnology pan stat1 sc 346
    Fig. 1. <t>STAT1</t> 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.
    Pan Stat1 Sc 346, 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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    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.

    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

    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.

    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

    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.

    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

    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.

    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

    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.

    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