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HMPV inhibits <t>STAT1</t> phosphorylation of Vero cells only in infected cells. (A) Vero cells were infected for 24 h with HMPV at MOI of 1 in 6-well plates. Cells were lysed with RIPA buffer, run on 4-15% bis-tris gel, transferred to nitrocellulose membrane,
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1) Product Images from "Human metapneumovirus small hydrophobic (SH) protein downregulates type I IFN pathway signaling by affecting STAT1 expression and phosphorylation"

Article Title: Human metapneumovirus small hydrophobic (SH) protein downregulates type I IFN pathway signaling by affecting STAT1 expression and phosphorylation

Journal: Virology

doi: 10.1016/j.virol.2016.04.022

HMPV inhibits STAT1 phosphorylation of Vero cells only in infected cells. (A) Vero cells were infected for 24 h with HMPV at MOI of 1 in 6-well plates. Cells were lysed with RIPA buffer, run on 4-15% bis-tris gel, transferred to nitrocellulose membrane,
Figure Legend Snippet: HMPV inhibits STAT1 phosphorylation of Vero cells only in infected cells. (A) Vero cells were infected for 24 h with HMPV at MOI of 1 in 6-well plates. Cells were lysed with RIPA buffer, run on 4-15% bis-tris gel, transferred to nitrocellulose membrane,

Techniques Used: Infection

Type I IFN secreted by infected BEAS-2B cells inhibits STAT1 phosphorylation in neighboring uninfected cells. (A) BEAS-2B cells were infected with HMPV for 24 h at an MOI of 1 in 6-well plates, then stained with polyclonal anti-HMPV antibody and gated
Figure Legend Snippet: Type I IFN secreted by infected BEAS-2B cells inhibits STAT1 phosphorylation in neighboring uninfected cells. (A) BEAS-2B cells were infected with HMPV for 24 h at an MOI of 1 in 6-well plates, then stained with polyclonal anti-HMPV antibody and gated

Techniques Used: Infection, Staining

HMPV inhibits STAT1 phosphorylation in BEAS-2B cells. (A) BEAS-2B cells were infected for 24 h with HMPV at an MOI of 1 in 6-well plates. Cells were lysed with RIPA buffer, run on 4-15% bis-tris gel, transferred to nitrocellulose membrane, and probed
Figure Legend Snippet: HMPV inhibits STAT1 phosphorylation in BEAS-2B cells. (A) BEAS-2B cells were infected for 24 h with HMPV at an MOI of 1 in 6-well plates. Cells were lysed with RIPA buffer, run on 4-15% bis-tris gel, transferred to nitrocellulose membrane, and probed

Techniques Used: Infection

Transient expression of HMPV SH protein inhibits STAT1 phosphorylation. (A) Vero cells transfected with HMPV GFP-fusion proteins were analyzed compared to untransfected cells in the same culture using flow cytometry. (B) Transfected cells were analyzed
Figure Legend Snippet: Transient expression of HMPV SH protein inhibits STAT1 phosphorylation. (A) Vero cells transfected with HMPV GFP-fusion proteins were analyzed compared to untransfected cells in the same culture using flow cytometry. (B) Transfected cells were analyzed

Techniques Used: Expressing, Transfection, Flow Cytometry, Cytometry

2) Product Images from "Human metapneumovirus small hydrophobic (SH) protein downregulates type I IFN pathway signaling by affecting STAT1 expression and phosphorylation"

Article Title: Human metapneumovirus small hydrophobic (SH) protein downregulates type I IFN pathway signaling by affecting STAT1 expression and phosphorylation

Journal: Virology

doi: 10.1016/j.virol.2016.04.022

HMPV inhibits STAT1 phosphorylation of Vero cells only in infected cells. (A) Vero cells were infected for 24 h with HMPV at MOI of 1 in 6-well plates. Cells were lysed with RIPA buffer, run on 4-15% bis-tris gel, transferred to nitrocellulose membrane,
Figure Legend Snippet: HMPV inhibits STAT1 phosphorylation of Vero cells only in infected cells. (A) Vero cells were infected for 24 h with HMPV at MOI of 1 in 6-well plates. Cells were lysed with RIPA buffer, run on 4-15% bis-tris gel, transferred to nitrocellulose membrane,

Techniques Used: Infection

Type I IFN secreted by infected BEAS-2B cells inhibits STAT1 phosphorylation in neighboring uninfected cells. (A) BEAS-2B cells were infected with HMPV for 24 h at an MOI of 1 in 6-well plates, then stained with polyclonal anti-HMPV antibody and gated
Figure Legend Snippet: Type I IFN secreted by infected BEAS-2B cells inhibits STAT1 phosphorylation in neighboring uninfected cells. (A) BEAS-2B cells were infected with HMPV for 24 h at an MOI of 1 in 6-well plates, then stained with polyclonal anti-HMPV antibody and gated

Techniques Used: Infection, Staining

HMPV inhibits STAT1 phosphorylation in BEAS-2B cells. (A) BEAS-2B cells were infected for 24 h with HMPV at an MOI of 1 in 6-well plates. Cells were lysed with RIPA buffer, run on 4-15% bis-tris gel, transferred to nitrocellulose membrane, and probed
Figure Legend Snippet: HMPV inhibits STAT1 phosphorylation in BEAS-2B cells. (A) BEAS-2B cells were infected for 24 h with HMPV at an MOI of 1 in 6-well plates. Cells were lysed with RIPA buffer, run on 4-15% bis-tris gel, transferred to nitrocellulose membrane, and probed

Techniques Used: Infection

Transient expression of HMPV SH protein inhibits STAT1 phosphorylation. (A) Vero cells transfected with HMPV GFP-fusion proteins were analyzed compared to untransfected cells in the same culture using flow cytometry. (B) Transfected cells were analyzed
Figure Legend Snippet: Transient expression of HMPV SH protein inhibits STAT1 phosphorylation. (A) Vero cells transfected with HMPV GFP-fusion proteins were analyzed compared to untransfected cells in the same culture using flow cytometry. (B) Transfected cells were analyzed

Techniques Used: Expressing, Transfection, Flow Cytometry, Cytometry

3) Product Images from "Human metapneumovirus small hydrophobic (SH) protein downregulates type I IFN pathway signaling by affecting STAT1 expression and phosphorylation"

Article Title: Human metapneumovirus small hydrophobic (SH) protein downregulates type I IFN pathway signaling by affecting STAT1 expression and phosphorylation

Journal: Virology

doi: 10.1016/j.virol.2016.04.022

HMPV inhibits STAT1 phosphorylation of Vero cells only in infected cells. (A) Vero cells were infected for 24 h with HMPV at MOI of 1 in 6-well plates. Cells were lysed with RIPA buffer, run on 4-15% bis-tris gel, transferred to nitrocellulose membrane,
Figure Legend Snippet: HMPV inhibits STAT1 phosphorylation of Vero cells only in infected cells. (A) Vero cells were infected for 24 h with HMPV at MOI of 1 in 6-well plates. Cells were lysed with RIPA buffer, run on 4-15% bis-tris gel, transferred to nitrocellulose membrane,

Techniques Used: Infection

Type I IFN secreted by infected BEAS-2B cells inhibits STAT1 phosphorylation in neighboring uninfected cells. (A) BEAS-2B cells were infected with HMPV for 24 h at an MOI of 1 in 6-well plates, then stained with polyclonal anti-HMPV antibody and gated
Figure Legend Snippet: Type I IFN secreted by infected BEAS-2B cells inhibits STAT1 phosphorylation in neighboring uninfected cells. (A) BEAS-2B cells were infected with HMPV for 24 h at an MOI of 1 in 6-well plates, then stained with polyclonal anti-HMPV antibody and gated

Techniques Used: Infection, Staining

HMPV inhibits STAT1 phosphorylation in BEAS-2B cells. (A) BEAS-2B cells were infected for 24 h with HMPV at an MOI of 1 in 6-well plates. Cells were lysed with RIPA buffer, run on 4-15% bis-tris gel, transferred to nitrocellulose membrane, and probed
Figure Legend Snippet: HMPV inhibits STAT1 phosphorylation in BEAS-2B cells. (A) BEAS-2B cells were infected for 24 h with HMPV at an MOI of 1 in 6-well plates. Cells were lysed with RIPA buffer, run on 4-15% bis-tris gel, transferred to nitrocellulose membrane, and probed

Techniques Used: Infection

Transient expression of HMPV SH protein inhibits STAT1 phosphorylation. (A) Vero cells transfected with HMPV GFP-fusion proteins were analyzed compared to untransfected cells in the same culture using flow cytometry. (B) Transfected cells were analyzed
Figure Legend Snippet: Transient expression of HMPV SH protein inhibits STAT1 phosphorylation. (A) Vero cells transfected with HMPV GFP-fusion proteins were analyzed compared to untransfected cells in the same culture using flow cytometry. (B) Transfected cells were analyzed

Techniques Used: Expressing, Transfection, Flow Cytometry, Cytometry

4) Product Images from "Human metapneumovirus small hydrophobic (SH) protein downregulates type I IFN pathway signaling by affecting STAT1 expression and phosphorylation"

Article Title: Human metapneumovirus small hydrophobic (SH) protein downregulates type I IFN pathway signaling by affecting STAT1 expression and phosphorylation

Journal: Virology

doi: 10.1016/j.virol.2016.04.022

HMPV inhibits STAT1 phosphorylation of Vero cells only in infected cells. (A) Vero cells were infected for 24 h with HMPV at MOI of 1 in 6-well plates. Cells were lysed with RIPA buffer, run on 4-15% bis-tris gel, transferred to nitrocellulose membrane,
Figure Legend Snippet: HMPV inhibits STAT1 phosphorylation of Vero cells only in infected cells. (A) Vero cells were infected for 24 h with HMPV at MOI of 1 in 6-well plates. Cells were lysed with RIPA buffer, run on 4-15% bis-tris gel, transferred to nitrocellulose membrane,

Techniques Used: Infection

Type I IFN secreted by infected BEAS-2B cells inhibits STAT1 phosphorylation in neighboring uninfected cells. (A) BEAS-2B cells were infected with HMPV for 24 h at an MOI of 1 in 6-well plates, then stained with polyclonal anti-HMPV antibody and gated
Figure Legend Snippet: Type I IFN secreted by infected BEAS-2B cells inhibits STAT1 phosphorylation in neighboring uninfected cells. (A) BEAS-2B cells were infected with HMPV for 24 h at an MOI of 1 in 6-well plates, then stained with polyclonal anti-HMPV antibody and gated

Techniques Used: Infection, Staining

HMPV inhibits STAT1 phosphorylation in BEAS-2B cells. (A) BEAS-2B cells were infected for 24 h with HMPV at an MOI of 1 in 6-well plates. Cells were lysed with RIPA buffer, run on 4-15% bis-tris gel, transferred to nitrocellulose membrane, and probed
Figure Legend Snippet: HMPV inhibits STAT1 phosphorylation in BEAS-2B cells. (A) BEAS-2B cells were infected for 24 h with HMPV at an MOI of 1 in 6-well plates. Cells were lysed with RIPA buffer, run on 4-15% bis-tris gel, transferred to nitrocellulose membrane, and probed

Techniques Used: Infection

Transient expression of HMPV SH protein inhibits STAT1 phosphorylation. (A) Vero cells transfected with HMPV GFP-fusion proteins were analyzed compared to untransfected cells in the same culture using flow cytometry. (B) Transfected cells were analyzed
Figure Legend Snippet: Transient expression of HMPV SH protein inhibits STAT1 phosphorylation. (A) Vero cells transfected with HMPV GFP-fusion proteins were analyzed compared to untransfected cells in the same culture using flow cytometry. (B) Transfected cells were analyzed

Techniques Used: Expressing, Transfection, Flow Cytometry, Cytometry

5) Product Images from "NS5 of Dengue Virus Mediates STAT2 Binding and Degradation ▿"

Article Title: NS5 of Dengue Virus Mediates STAT2 Binding and Degradation ▿

Journal: Journal of Virology

doi: 10.1128/JVI.02188-08

Truncations in the DENV NS5 protein affect its ability to associate with STAT2, decrease STAT2 levels, and inhibit IFN signaling. (A) 293T cells were transfected with the indicated constructs. Numbering refers to the glycine start position within the NS5 protein. All numbered forms of NS5 are expressed within the context of the E-Ub-NS5-GFP construct (i.e., 10-900, NS5 residues 10 to 900 fused to the C terminus of the E-Ub cassette). Twenty-four hours posttransfection, cells were sorted for GFP-positive cells using FACS, lysed, and examined by Western blotting using STAT2, GFP, and tubulin antibodies. (B) 293T cells were cotransfected with the indicated plasmids (the numbering system is identical to that described in A), STAT1-FLAG, and STAT2-FLAG. In order to detect STAT2 binding by NS5, STAT2-FLAG plasmid was transfected in excess with respect to E-Ub-NS5-GFP plasmids, resulting in a not-detectable degradation of STAT2-FLAG. Lysates were immunoprecipitated with a polyclonal GFP antibody (pGFP), and Western blots were performed using FLAG, GFP, and GAPDH antibodies. TCE, total cell extracts. (C) 293T cells were transfected with the ISRE-54-CAT reporter, a constitutively expressing firefly luciferase plasmid, and the indicated HA-tagged viral protein. Twenty-four hours posttransfection, cells were treated with 1,000 U/ml of type I IFN. Twenty-four hours posttreatment, cells were lysed and measured for CAT and luciferase activity. Data are represented with the standard deviations from three independent experiments. Samples showing P values of less than 0.05 compared with the empty control sample are indicated.
Figure Legend Snippet: Truncations in the DENV NS5 protein affect its ability to associate with STAT2, decrease STAT2 levels, and inhibit IFN signaling. (A) 293T cells were transfected with the indicated constructs. Numbering refers to the glycine start position within the NS5 protein. All numbered forms of NS5 are expressed within the context of the E-Ub-NS5-GFP construct (i.e., 10-900, NS5 residues 10 to 900 fused to the C terminus of the E-Ub cassette). Twenty-four hours posttransfection, cells were sorted for GFP-positive cells using FACS, lysed, and examined by Western blotting using STAT2, GFP, and tubulin antibodies. (B) 293T cells were cotransfected with the indicated plasmids (the numbering system is identical to that described in A), STAT1-FLAG, and STAT2-FLAG. In order to detect STAT2 binding by NS5, STAT2-FLAG plasmid was transfected in excess with respect to E-Ub-NS5-GFP plasmids, resulting in a not-detectable degradation of STAT2-FLAG. Lysates were immunoprecipitated with a polyclonal GFP antibody (pGFP), and Western blots were performed using FLAG, GFP, and GAPDH antibodies. TCE, total cell extracts. (C) 293T cells were transfected with the ISRE-54-CAT reporter, a constitutively expressing firefly luciferase plasmid, and the indicated HA-tagged viral protein. Twenty-four hours posttransfection, cells were treated with 1,000 U/ml of type I IFN. Twenty-four hours posttreatment, cells were lysed and measured for CAT and luciferase activity. Data are represented with the standard deviations from three independent experiments. Samples showing P values of less than 0.05 compared with the empty control sample are indicated.

Techniques Used: Transfection, Construct, FACS, Western Blot, Binding Assay, Plasmid Preparation, Immunoprecipitation, Expressing, Luciferase, Activity Assay

Inhibitors of the ubiquitin-proteasome pathway prevent STAT2 degradation by DENV NS5. (A) wtVero or Vero cells stably expressing the DEN1 replicon were treated with the indicated amounts of MG132. Sixteen hours posttreatment, cells were lysed and examined for ubiquitin, STAT2, STAT1, NS5, and GAPDH levels via Western blotting. (B) STAT2-deficient U6A cells were transfected with HA-ubiquitin, STAT2-FLAG, STAT1-GFP, NS2b-3, and either E- clv NS5-HA or an empty vector plasmid. Cells were then treated with lactacystin for 8 h and subsequently lysed and examined by Western blotting using ubiquitin, STAT2, STAT1, HA, and tubulin antibodies. (C) 293T cells were cotransfected with NS2b-3-HA and the plasmids indicated at the top. Ten hours posttransfection, cells were treated with the indicated amounts of lactacystin. Twenty-four hours posttransfection, cells were sorted for GFP-positive cells by FACS, lysed, and examined by Western blotting using ubiquitin-, GFP-, HA-, and GAPDH-specific antibodies. (D) STAT2-deficient U6A cells were transfected with 0.1 or 1 μg of FLAG-STAT2-FLAG, 0.1 μg NS2b-3, and either 1 μg E- clv NS5-HA or an empty vector plasmid. Cells were then lysed and examined by Western blotting using FLAG antibody and long-term film exposure to detect any additional low-intensity bands. The arrow indicates the expected size of FLAG-STAT2-FLAG. The asterisk marks a nonspecific band running at the same mobility of FLAG-STAT2-FLAG. (E) 293T cells were cotransfected with NS2b-3 and the plasmids indicated at the top. Twenty-four hours posttransfection, cells were sorted for GFP-positive cells by FACS, lysed, and examined via Western blotting using STAT2-, GFP-, HA-, FLAG-, and GAPDH-specific antibodies.
Figure Legend Snippet: Inhibitors of the ubiquitin-proteasome pathway prevent STAT2 degradation by DENV NS5. (A) wtVero or Vero cells stably expressing the DEN1 replicon were treated with the indicated amounts of MG132. Sixteen hours posttreatment, cells were lysed and examined for ubiquitin, STAT2, STAT1, NS5, and GAPDH levels via Western blotting. (B) STAT2-deficient U6A cells were transfected with HA-ubiquitin, STAT2-FLAG, STAT1-GFP, NS2b-3, and either E- clv NS5-HA or an empty vector plasmid. Cells were then treated with lactacystin for 8 h and subsequently lysed and examined by Western blotting using ubiquitin, STAT2, STAT1, HA, and tubulin antibodies. (C) 293T cells were cotransfected with NS2b-3-HA and the plasmids indicated at the top. Ten hours posttransfection, cells were treated with the indicated amounts of lactacystin. Twenty-four hours posttransfection, cells were sorted for GFP-positive cells by FACS, lysed, and examined by Western blotting using ubiquitin-, GFP-, HA-, and GAPDH-specific antibodies. (D) STAT2-deficient U6A cells were transfected with 0.1 or 1 μg of FLAG-STAT2-FLAG, 0.1 μg NS2b-3, and either 1 μg E- clv NS5-HA or an empty vector plasmid. Cells were then lysed and examined by Western blotting using FLAG antibody and long-term film exposure to detect any additional low-intensity bands. The arrow indicates the expected size of FLAG-STAT2-FLAG. The asterisk marks a nonspecific band running at the same mobility of FLAG-STAT2-FLAG. (E) 293T cells were cotransfected with NS2b-3 and the plasmids indicated at the top. Twenty-four hours posttransfection, cells were sorted for GFP-positive cells by FACS, lysed, and examined via Western blotting using STAT2-, GFP-, HA-, FLAG-, and GAPDH-specific antibodies.

Techniques Used: Stable Transfection, Expressing, Western Blot, Transfection, Plasmid Preparation, FACS

DENV NS5 interacts with STAT2. (A) 293T cells were cotransfected with plasmids expressing FLAG-tagged STAT1 (STAT1-FLAG) and STAT2 (STAT2-FLAG) and empty plasmid (empty) or plasmid expressing HA-tagged DENV NS5 (NS5-HA), DENV core (CORE-HA), or NiV-V (HA-NiV-V) proteins. Lysates were then immunoprecipitated with anti-HA antibody (IP HA), and Western blotting was performed using anti-HA and anti-FLAG antibodies. Asterisks mark the heavy and light chains from the HA antibody. (B) 293T cells were cotransfected with plasmids expressing NS5-HA and either STAT1-FLAG or STAT2-FLAG. Lysates were then immunoprecipitated with anti-FLAG antibody (IP FLAG), and Western blotting was performed using anti-HA and anti-FLAG antibodies. TCE, total cell extracts were subjected to Western blotting using anti-HA, anti-FLAG, and anti-GAPDH antibodies.
Figure Legend Snippet: DENV NS5 interacts with STAT2. (A) 293T cells were cotransfected with plasmids expressing FLAG-tagged STAT1 (STAT1-FLAG) and STAT2 (STAT2-FLAG) and empty plasmid (empty) or plasmid expressing HA-tagged DENV NS5 (NS5-HA), DENV core (CORE-HA), or NiV-V (HA-NiV-V) proteins. Lysates were then immunoprecipitated with anti-HA antibody (IP HA), and Western blotting was performed using anti-HA and anti-FLAG antibodies. Asterisks mark the heavy and light chains from the HA antibody. (B) 293T cells were cotransfected with plasmids expressing NS5-HA and either STAT1-FLAG or STAT2-FLAG. Lysates were then immunoprecipitated with anti-FLAG antibody (IP FLAG), and Western blotting was performed using anti-HA and anti-FLAG antibodies. TCE, total cell extracts were subjected to Western blotting using anti-HA, anti-FLAG, and anti-GAPDH antibodies.

Techniques Used: Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot

Expression of a precursor form of DENV NS5 cleaved by the DENV protease results in reduced STAT2 levels. (A) 293T cells were transfected with the indicated constructs. Twenty-four hours posttransfection, cells were sorted for GFP-positive cells by FACS, subsequently lysed, and examined via Western blotting using GFP-, HA-, STAT1-, STAT2-, and GAPDH-specific antibodies. Schematics of the transfected constructs are shown at the bottom. ORFs that contain an “sp” at the N terminus have a signal peptide which directs the entire polyprotein to the surface of the ER for translation. Black arrows and red arrows indicate cleavage sites for cellular and the DEN2 viral proteases, respectively. The DEN2 active protease is highlighted in red. Densitometry analysis of the levels of STAT2 and NS5 are included on the far right, and levels are calculated relative to the levels in lane 1, with a value of 1 in the case of NS5 levels being indicative of no detection (background levels). (B) Same as above (A). Mutation of the DENV protease recognition site at the N terminus of NS5 is indicated by the blue dashes. The DENV protease labeled in blue indicates a serine-to-alanine mutation within the catalytic site of the DENV protease.
Figure Legend Snippet: Expression of a precursor form of DENV NS5 cleaved by the DENV protease results in reduced STAT2 levels. (A) 293T cells were transfected with the indicated constructs. Twenty-four hours posttransfection, cells were sorted for GFP-positive cells by FACS, subsequently lysed, and examined via Western blotting using GFP-, HA-, STAT1-, STAT2-, and GAPDH-specific antibodies. Schematics of the transfected constructs are shown at the bottom. ORFs that contain an “sp” at the N terminus have a signal peptide which directs the entire polyprotein to the surface of the ER for translation. Black arrows and red arrows indicate cleavage sites for cellular and the DEN2 viral proteases, respectively. The DEN2 active protease is highlighted in red. Densitometry analysis of the levels of STAT2 and NS5 are included on the far right, and levels are calculated relative to the levels in lane 1, with a value of 1 in the case of NS5 levels being indicative of no detection (background levels). (B) Same as above (A). Mutation of the DENV protease recognition site at the N terminus of NS5 is indicated by the blue dashes. The DENV protease labeled in blue indicates a serine-to-alanine mutation within the catalytic site of the DENV protease.

Techniques Used: Expressing, Transfection, Construct, FACS, Western Blot, Mutagenesis, Labeling

Cleaved NS5 is sufficient for reduced STAT2 levels and does not require an N-terminal glycine. (A) 293T cells were transfected with the indicated constructs. Twenty-four hours posttransfection, cells were sorted for GFP-positive cells by FACS, subsequently lysed, subjected to 4-to-20% SDS-polyacrylamide gel electrophoresis, and examined via Western blotting using GFP-, HA-, STAT1-, STAT2-, and GAPDH-specific antibodies. The TEV protease and its cleavage site are indicated by the green text and arrow, respectively. Cleavage sequences targeted by endogenous deubiquitinases are noted by a yellow arrow. Densitometry analyses of the levels of STAT2 and NS5 are included at the bottom, and levels were calculated relative to the levels in lane 1, with a value of 1 in the case of NS5 levels being indicative of no detection (background levels). (B) Same as above (A) except that lysates were run on a 7.5% SDS-polyacrylamide gel and subsequently analyzed by Western blotting using GFP-, STAT2-, and tubulin-specific antibodies.
Figure Legend Snippet: Cleaved NS5 is sufficient for reduced STAT2 levels and does not require an N-terminal glycine. (A) 293T cells were transfected with the indicated constructs. Twenty-four hours posttransfection, cells were sorted for GFP-positive cells by FACS, subsequently lysed, subjected to 4-to-20% SDS-polyacrylamide gel electrophoresis, and examined via Western blotting using GFP-, HA-, STAT1-, STAT2-, and GAPDH-specific antibodies. The TEV protease and its cleavage site are indicated by the green text and arrow, respectively. Cleavage sequences targeted by endogenous deubiquitinases are noted by a yellow arrow. Densitometry analyses of the levels of STAT2 and NS5 are included at the bottom, and levels were calculated relative to the levels in lane 1, with a value of 1 in the case of NS5 levels being indicative of no detection (background levels). (B) Same as above (A) except that lysates were run on a 7.5% SDS-polyacrylamide gel and subsequently analyzed by Western blotting using GFP-, STAT2-, and tubulin-specific antibodies.

Techniques Used: Transfection, Construct, FACS, Polyacrylamide Gel Electrophoresis, Western Blot

6) Product Images from "NS5 of Dengue Virus Mediates STAT2 Binding and Degradation ▿"

Article Title: NS5 of Dengue Virus Mediates STAT2 Binding and Degradation ▿

Journal: Journal of Virology

doi: 10.1128/JVI.02188-08

Truncations in the DENV NS5 protein affect its ability to associate with STAT2, decrease STAT2 levels, and inhibit IFN signaling. (A) 293T cells were transfected with the indicated constructs. Numbering refers to the glycine start position within the NS5 protein. All numbered forms of NS5 are expressed within the context of the E-Ub-NS5-GFP construct (i.e., 10-900, NS5 residues 10 to 900 fused to the C terminus of the E-Ub cassette). Twenty-four hours posttransfection, cells were sorted for GFP-positive cells using FACS, lysed, and examined by Western blotting using STAT2, GFP, and tubulin antibodies. (B) 293T cells were cotransfected with the indicated plasmids (the numbering system is identical to that described in A), STAT1-FLAG, and STAT2-FLAG. In order to detect STAT2 binding by NS5, STAT2-FLAG plasmid was transfected in excess with respect to E-Ub-NS5-GFP plasmids, resulting in a not-detectable degradation of STAT2-FLAG. Lysates were immunoprecipitated with a polyclonal GFP antibody (pGFP), and Western blots were performed using FLAG, GFP, and GAPDH antibodies. TCE, total cell extracts. (C) 293T cells were transfected with the ISRE-54-CAT reporter, a constitutively expressing firefly luciferase plasmid, and the indicated HA-tagged viral protein. Twenty-four hours posttransfection, cells were treated with 1,000 U/ml of type I IFN. Twenty-four hours posttreatment, cells were lysed and measured for CAT and luciferase activity. Data are represented with the standard deviations from three independent experiments. Samples showing P values of less than 0.05 compared with the empty control sample are indicated.
Figure Legend Snippet: Truncations in the DENV NS5 protein affect its ability to associate with STAT2, decrease STAT2 levels, and inhibit IFN signaling. (A) 293T cells were transfected with the indicated constructs. Numbering refers to the glycine start position within the NS5 protein. All numbered forms of NS5 are expressed within the context of the E-Ub-NS5-GFP construct (i.e., 10-900, NS5 residues 10 to 900 fused to the C terminus of the E-Ub cassette). Twenty-four hours posttransfection, cells were sorted for GFP-positive cells using FACS, lysed, and examined by Western blotting using STAT2, GFP, and tubulin antibodies. (B) 293T cells were cotransfected with the indicated plasmids (the numbering system is identical to that described in A), STAT1-FLAG, and STAT2-FLAG. In order to detect STAT2 binding by NS5, STAT2-FLAG plasmid was transfected in excess with respect to E-Ub-NS5-GFP plasmids, resulting in a not-detectable degradation of STAT2-FLAG. Lysates were immunoprecipitated with a polyclonal GFP antibody (pGFP), and Western blots were performed using FLAG, GFP, and GAPDH antibodies. TCE, total cell extracts. (C) 293T cells were transfected with the ISRE-54-CAT reporter, a constitutively expressing firefly luciferase plasmid, and the indicated HA-tagged viral protein. Twenty-four hours posttransfection, cells were treated with 1,000 U/ml of type I IFN. Twenty-four hours posttreatment, cells were lysed and measured for CAT and luciferase activity. Data are represented with the standard deviations from three independent experiments. Samples showing P values of less than 0.05 compared with the empty control sample are indicated.

Techniques Used: Transfection, Construct, FACS, Western Blot, Binding Assay, Plasmid Preparation, Immunoprecipitation, Expressing, Luciferase, Activity Assay

Inhibitors of the ubiquitin-proteasome pathway prevent STAT2 degradation by DENV NS5. (A) wtVero or Vero cells stably expressing the DEN1 replicon were treated with the indicated amounts of MG132. Sixteen hours posttreatment, cells were lysed and examined for ubiquitin, STAT2, STAT1, NS5, and GAPDH levels via Western blotting. (B) STAT2-deficient U6A cells were transfected with HA-ubiquitin, STAT2-FLAG, STAT1-GFP, NS2b-3, and either E- clv NS5-HA or an empty vector plasmid. Cells were then treated with lactacystin for 8 h and subsequently lysed and examined by Western blotting using ubiquitin, STAT2, STAT1, HA, and tubulin antibodies. (C) 293T cells were cotransfected with NS2b-3-HA and the plasmids indicated at the top. Ten hours posttransfection, cells were treated with the indicated amounts of lactacystin. Twenty-four hours posttransfection, cells were sorted for GFP-positive cells by FACS, lysed, and examined by Western blotting using ubiquitin-, GFP-, HA-, and GAPDH-specific antibodies. (D) STAT2-deficient U6A cells were transfected with 0.1 or 1 μg of FLAG-STAT2-FLAG, 0.1 μg NS2b-3, and either 1 μg E- clv NS5-HA or an empty vector plasmid. Cells were then lysed and examined by Western blotting using FLAG antibody and long-term film exposure to detect any additional low-intensity bands. The arrow indicates the expected size of FLAG-STAT2-FLAG. The asterisk marks a nonspecific band running at the same mobility of FLAG-STAT2-FLAG. (E) 293T cells were cotransfected with NS2b-3 and the plasmids indicated at the top. Twenty-four hours posttransfection, cells were sorted for GFP-positive cells by FACS, lysed, and examined via Western blotting using STAT2-, GFP-, HA-, FLAG-, and GAPDH-specific antibodies.
Figure Legend Snippet: Inhibitors of the ubiquitin-proteasome pathway prevent STAT2 degradation by DENV NS5. (A) wtVero or Vero cells stably expressing the DEN1 replicon were treated with the indicated amounts of MG132. Sixteen hours posttreatment, cells were lysed and examined for ubiquitin, STAT2, STAT1, NS5, and GAPDH levels via Western blotting. (B) STAT2-deficient U6A cells were transfected with HA-ubiquitin, STAT2-FLAG, STAT1-GFP, NS2b-3, and either E- clv NS5-HA or an empty vector plasmid. Cells were then treated with lactacystin for 8 h and subsequently lysed and examined by Western blotting using ubiquitin, STAT2, STAT1, HA, and tubulin antibodies. (C) 293T cells were cotransfected with NS2b-3-HA and the plasmids indicated at the top. Ten hours posttransfection, cells were treated with the indicated amounts of lactacystin. Twenty-four hours posttransfection, cells were sorted for GFP-positive cells by FACS, lysed, and examined by Western blotting using ubiquitin-, GFP-, HA-, and GAPDH-specific antibodies. (D) STAT2-deficient U6A cells were transfected with 0.1 or 1 μg of FLAG-STAT2-FLAG, 0.1 μg NS2b-3, and either 1 μg E- clv NS5-HA or an empty vector plasmid. Cells were then lysed and examined by Western blotting using FLAG antibody and long-term film exposure to detect any additional low-intensity bands. The arrow indicates the expected size of FLAG-STAT2-FLAG. The asterisk marks a nonspecific band running at the same mobility of FLAG-STAT2-FLAG. (E) 293T cells were cotransfected with NS2b-3 and the plasmids indicated at the top. Twenty-four hours posttransfection, cells were sorted for GFP-positive cells by FACS, lysed, and examined via Western blotting using STAT2-, GFP-, HA-, FLAG-, and GAPDH-specific antibodies.

Techniques Used: Stable Transfection, Expressing, Western Blot, Transfection, Plasmid Preparation, FACS

DENV NS5 interacts with STAT2. (A) 293T cells were cotransfected with plasmids expressing FLAG-tagged STAT1 (STAT1-FLAG) and STAT2 (STAT2-FLAG) and empty plasmid (empty) or plasmid expressing HA-tagged DENV NS5 (NS5-HA), DENV core (CORE-HA), or NiV-V (HA-NiV-V) proteins. Lysates were then immunoprecipitated with anti-HA antibody (IP HA), and Western blotting was performed using anti-HA and anti-FLAG antibodies. Asterisks mark the heavy and light chains from the HA antibody. (B) 293T cells were cotransfected with plasmids expressing NS5-HA and either STAT1-FLAG or STAT2-FLAG. Lysates were then immunoprecipitated with anti-FLAG antibody (IP FLAG), and Western blotting was performed using anti-HA and anti-FLAG antibodies. TCE, total cell extracts were subjected to Western blotting using anti-HA, anti-FLAG, and anti-GAPDH antibodies.
Figure Legend Snippet: DENV NS5 interacts with STAT2. (A) 293T cells were cotransfected with plasmids expressing FLAG-tagged STAT1 (STAT1-FLAG) and STAT2 (STAT2-FLAG) and empty plasmid (empty) or plasmid expressing HA-tagged DENV NS5 (NS5-HA), DENV core (CORE-HA), or NiV-V (HA-NiV-V) proteins. Lysates were then immunoprecipitated with anti-HA antibody (IP HA), and Western blotting was performed using anti-HA and anti-FLAG antibodies. Asterisks mark the heavy and light chains from the HA antibody. (B) 293T cells were cotransfected with plasmids expressing NS5-HA and either STAT1-FLAG or STAT2-FLAG. Lysates were then immunoprecipitated with anti-FLAG antibody (IP FLAG), and Western blotting was performed using anti-HA and anti-FLAG antibodies. TCE, total cell extracts were subjected to Western blotting using anti-HA, anti-FLAG, and anti-GAPDH antibodies.

Techniques Used: Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot

Expression of a precursor form of DENV NS5 cleaved by the DENV protease results in reduced STAT2 levels. (A) 293T cells were transfected with the indicated constructs. Twenty-four hours posttransfection, cells were sorted for GFP-positive cells by FACS, subsequently lysed, and examined via Western blotting using GFP-, HA-, STAT1-, STAT2-, and GAPDH-specific antibodies. Schematics of the transfected constructs are shown at the bottom. ORFs that contain an “sp” at the N terminus have a signal peptide which directs the entire polyprotein to the surface of the ER for translation. Black arrows and red arrows indicate cleavage sites for cellular and the DEN2 viral proteases, respectively. The DEN2 active protease is highlighted in red. Densitometry analysis of the levels of STAT2 and NS5 are included on the far right, and levels are calculated relative to the levels in lane 1, with a value of 1 in the case of NS5 levels being indicative of no detection (background levels). (B) Same as above (A). Mutation of the DENV protease recognition site at the N terminus of NS5 is indicated by the blue dashes. The DENV protease labeled in blue indicates a serine-to-alanine mutation within the catalytic site of the DENV protease.
Figure Legend Snippet: Expression of a precursor form of DENV NS5 cleaved by the DENV protease results in reduced STAT2 levels. (A) 293T cells were transfected with the indicated constructs. Twenty-four hours posttransfection, cells were sorted for GFP-positive cells by FACS, subsequently lysed, and examined via Western blotting using GFP-, HA-, STAT1-, STAT2-, and GAPDH-specific antibodies. Schematics of the transfected constructs are shown at the bottom. ORFs that contain an “sp” at the N terminus have a signal peptide which directs the entire polyprotein to the surface of the ER for translation. Black arrows and red arrows indicate cleavage sites for cellular and the DEN2 viral proteases, respectively. The DEN2 active protease is highlighted in red. Densitometry analysis of the levels of STAT2 and NS5 are included on the far right, and levels are calculated relative to the levels in lane 1, with a value of 1 in the case of NS5 levels being indicative of no detection (background levels). (B) Same as above (A). Mutation of the DENV protease recognition site at the N terminus of NS5 is indicated by the blue dashes. The DENV protease labeled in blue indicates a serine-to-alanine mutation within the catalytic site of the DENV protease.

Techniques Used: Expressing, Transfection, Construct, FACS, Western Blot, Mutagenesis, Labeling

Cleaved NS5 is sufficient for reduced STAT2 levels and does not require an N-terminal glycine. (A) 293T cells were transfected with the indicated constructs. Twenty-four hours posttransfection, cells were sorted for GFP-positive cells by FACS, subsequently lysed, subjected to 4-to-20% SDS-polyacrylamide gel electrophoresis, and examined via Western blotting using GFP-, HA-, STAT1-, STAT2-, and GAPDH-specific antibodies. The TEV protease and its cleavage site are indicated by the green text and arrow, respectively. Cleavage sequences targeted by endogenous deubiquitinases are noted by a yellow arrow. Densitometry analyses of the levels of STAT2 and NS5 are included at the bottom, and levels were calculated relative to the levels in lane 1, with a value of 1 in the case of NS5 levels being indicative of no detection (background levels). (B) Same as above (A) except that lysates were run on a 7.5% SDS-polyacrylamide gel and subsequently analyzed by Western blotting using GFP-, STAT2-, and tubulin-specific antibodies.
Figure Legend Snippet: Cleaved NS5 is sufficient for reduced STAT2 levels and does not require an N-terminal glycine. (A) 293T cells were transfected with the indicated constructs. Twenty-four hours posttransfection, cells were sorted for GFP-positive cells by FACS, subsequently lysed, subjected to 4-to-20% SDS-polyacrylamide gel electrophoresis, and examined via Western blotting using GFP-, HA-, STAT1-, STAT2-, and GAPDH-specific antibodies. The TEV protease and its cleavage site are indicated by the green text and arrow, respectively. Cleavage sequences targeted by endogenous deubiquitinases are noted by a yellow arrow. Densitometry analyses of the levels of STAT2 and NS5 are included at the bottom, and levels were calculated relative to the levels in lane 1, with a value of 1 in the case of NS5 levels being indicative of no detection (background levels). (B) Same as above (A) except that lysates were run on a 7.5% SDS-polyacrylamide gel and subsequently analyzed by Western blotting using GFP-, STAT2-, and tubulin-specific antibodies.

Techniques Used: Transfection, Construct, FACS, Polyacrylamide Gel Electrophoresis, Western Blot

7) Product Images from "Expression of RASSF1A, an epigenetically silenced tumor suppressor, overcomes resistance to apoptosis induction by interferons"

Article Title: Expression of RASSF1A, an epigenetically silenced tumor suppressor, overcomes resistance to apoptosis induction by interferons

Journal: Cancer research

doi: 10.1158/0008-5472.CAN-05-2303

Depletion of DNMT1 by oligonucleotide antisense overcomes resistance to IFN-induced apoptosis ACHN (A) and SK-RC-45 (B) cells were transfected daily with 40 nM DNMT1 AS or mismatch oligonucleotide (MM) or lipofectin transfection reagent only (L or Lipo). Every other day, 4h after the preceding transfection, cells were replated at 15 000 cells/cm 2 (ACHN) or 5000 cells/cm 2 (SK-RC-45) to keep confluencies optimal for transfection efficiency. A): Near-complete depletion of DNMT1 was achieved by day 4 of AS treatment and maintained to day 8 allowing for cell divisions in the absence of the maintenance DNA methyltransferase. Expression of stat1, stat2, and stat3 was not affected. After 6–8 days cells were plated at 5000 cells/cm 2 for IFN treatment 16 h later. Apoptosis was assessed by immunoblotting for cleaved caspase 3 after 48 hr of IFN (following 8 days of DNMT1 AS) and by TUNEL assay to detect cells containing fragmented DNA after 5 days of IFN (following 6 and 8 days of DNMT1 AS). Duration of DNMT1 depletion correlated with apoptosis induction by 50 U/ml IFN-α 2 or IFN-β as determined by TUNEL assay, while MM did not sensitize. B) DNMT1 AS (AS) at 40nM was sufficient for near-complete depletion of DNMT1 protein by day 4 in SK-RC-45 cells. Resistance to apoptosis induction by 500 U/ml of IFN-α 2 or IFN-β over 4 days was overcome by pretreatment with DNMT1 AS over 6 days. On TUNEL graphs the means and standard deviations (error bars) of independent experiments are displayed.
Figure Legend Snippet: Depletion of DNMT1 by oligonucleotide antisense overcomes resistance to IFN-induced apoptosis ACHN (A) and SK-RC-45 (B) cells were transfected daily with 40 nM DNMT1 AS or mismatch oligonucleotide (MM) or lipofectin transfection reagent only (L or Lipo). Every other day, 4h after the preceding transfection, cells were replated at 15 000 cells/cm 2 (ACHN) or 5000 cells/cm 2 (SK-RC-45) to keep confluencies optimal for transfection efficiency. A): Near-complete depletion of DNMT1 was achieved by day 4 of AS treatment and maintained to day 8 allowing for cell divisions in the absence of the maintenance DNA methyltransferase. Expression of stat1, stat2, and stat3 was not affected. After 6–8 days cells were plated at 5000 cells/cm 2 for IFN treatment 16 h later. Apoptosis was assessed by immunoblotting for cleaved caspase 3 after 48 hr of IFN (following 8 days of DNMT1 AS) and by TUNEL assay to detect cells containing fragmented DNA after 5 days of IFN (following 6 and 8 days of DNMT1 AS). Duration of DNMT1 depletion correlated with apoptosis induction by 50 U/ml IFN-α 2 or IFN-β as determined by TUNEL assay, while MM did not sensitize. B) DNMT1 AS (AS) at 40nM was sufficient for near-complete depletion of DNMT1 protein by day 4 in SK-RC-45 cells. Resistance to apoptosis induction by 500 U/ml of IFN-α 2 or IFN-β over 4 days was overcome by pretreatment with DNMT1 AS over 6 days. On TUNEL graphs the means and standard deviations (error bars) of independent experiments are displayed.

Techniques Used: Transfection, Expressing, TUNEL Assay

8) Product Images from "Activation of Type I and III Interferon Signalling Pathways Occurs in Lung Epithelial Cells Infected with Low Pathogenic Avian Influenza Viruses"

Article Title: Activation of Type I and III Interferon Signalling Pathways Occurs in Lung Epithelial Cells Infected with Low Pathogenic Avian Influenza Viruses

Journal: PLoS ONE

doi: 10.1371/journal.pone.0033732

Influenza virus induced changes in STAT1 signalling during virus infection. (A) A549 cells were infected with either the H1N1/WSN, H9N2, H5N2/F118 or H5N3 viruses or (B) the pH1N1/471, pH1N1/478 or pH1N1/527 virus using an MOI = 4. Then cells were harvested at between 0.2 and 18 hpi in SDS-PAGE boiling mix as described in methods . The proteins were transferred on to PVDF membranes by western blotting, and the membranes probed with the appropriate primary and secondary antibodies. The phosphorylated STAT-1 (pSTAT1) and total STAT-1(STAT1) are shown. (A) β-catenin or (B) β-actin provides a loading control. (C) A549 cells were either mock-infected or were infected with the H1N1/WSN, H5N2, H9N2, H5N3, pH1N1/471 or pH1N1/527 viruses using an MOI = 4. At 16 hpi the cells were labelled using anti-MX and goat anti-mouse conjugated to Alexa555. The stained cells were visualised using a Nikon Eclipse 80i Microscope at ×20 magnification using appropriate machine settings.
Figure Legend Snippet: Influenza virus induced changes in STAT1 signalling during virus infection. (A) A549 cells were infected with either the H1N1/WSN, H9N2, H5N2/F118 or H5N3 viruses or (B) the pH1N1/471, pH1N1/478 or pH1N1/527 virus using an MOI = 4. Then cells were harvested at between 0.2 and 18 hpi in SDS-PAGE boiling mix as described in methods . The proteins were transferred on to PVDF membranes by western blotting, and the membranes probed with the appropriate primary and secondary antibodies. The phosphorylated STAT-1 (pSTAT1) and total STAT-1(STAT1) are shown. (A) β-catenin or (B) β-actin provides a loading control. (C) A549 cells were either mock-infected or were infected with the H1N1/WSN, H5N2, H9N2, H5N3, pH1N1/471 or pH1N1/527 viruses using an MOI = 4. At 16 hpi the cells were labelled using anti-MX and goat anti-mouse conjugated to Alexa555. The stained cells were visualised using a Nikon Eclipse 80i Microscope at ×20 magnification using appropriate machine settings.

Techniques Used: Infection, SDS Page, Western Blot, Staining, Microscopy

9) Product Images from "Caveolin-2-deficient mice show increased sensitivity to endotoxemia"

Article Title: Caveolin-2-deficient mice show increased sensitivity to endotoxemia

Journal: Cell Cycle

doi: 10.4161/cc.10.13.16234

LPS-treated Cav-2 -/- mice exhibit increased levels of tyrosinephosphorylated STAT1 compared to LPS-treated WT and Cav-1 -/- mice. (A) The level of tyrosine-phosphorylated STAT1 (Tyr701) and total STAT1 were detected by immunoblot of colon lysates obtained
Figure Legend Snippet: LPS-treated Cav-2 -/- mice exhibit increased levels of tyrosinephosphorylated STAT1 compared to LPS-treated WT and Cav-1 -/- mice. (A) The level of tyrosine-phosphorylated STAT1 (Tyr701) and total STAT1 were detected by immunoblot of colon lysates obtained

Techniques Used: Mouse Assay

10) Product Images from "Pegylated IFN-? regulates hepatic gene expression through transient Jak/STAT activation"

Article Title: Pegylated IFN-? regulates hepatic gene expression through transient Jak/STAT activation

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI70408

pegIFN-α2b induced STAT1 phosphorylation and ISG expression in the liver.
Figure Legend Snippet: pegIFN-α2b induced STAT1 phosphorylation and ISG expression in the liver.

Techniques Used: Expressing

U-STAT1 does not induce ISGs. ( A ) Three clones with different expression levels of WT (WT cl1, WT cl2, and WT cl3) or mutated STAT1 (Y701F cl1, Y701F cl2, and Y701F cl3) were stimulated with IFN-α. STAT1-deficient U3A cells and STAT1 WT parental 2fTGH cells were used as controls. IFN-α induced STAT1 phosphorylation in 2fTGH and in all three WT clones. Actin is shown as a loading control. The cells were either untreated or treated for 15 minutes with 1,000 U/ml of IFN-α. WT cl1 and Y701F cl1 express STAT1 in an amount similar to that induced by 2fTGH cells treated for 24 hours with 1,000 U/ml of IFN-α. Shown are representative blots each from three independently performed experiments (black lanes separate blots that were derived from the same gel, but were noncontiguous). ( B ) IFN-α–induced OAS1 mRNA expression relative to GAPDH was assessed by qRT-PCR. Cells were treated with 1,000 U/ml IFN-α for 8 hours. Upregulation of OAS1 was found only in cells with WT STAT1 after IFN-α treatment. Expression of maximal amounts of Y701F-mutated STAT1 in U3A cells did not induce ISG expression. Shown are the mean values with SEM of three replicate experiments.
Figure Legend Snippet: U-STAT1 does not induce ISGs. ( A ) Three clones with different expression levels of WT (WT cl1, WT cl2, and WT cl3) or mutated STAT1 (Y701F cl1, Y701F cl2, and Y701F cl3) were stimulated with IFN-α. STAT1-deficient U3A cells and STAT1 WT parental 2fTGH cells were used as controls. IFN-α induced STAT1 phosphorylation in 2fTGH and in all three WT clones. Actin is shown as a loading control. The cells were either untreated or treated for 15 minutes with 1,000 U/ml of IFN-α. WT cl1 and Y701F cl1 express STAT1 in an amount similar to that induced by 2fTGH cells treated for 24 hours with 1,000 U/ml of IFN-α. Shown are representative blots each from three independently performed experiments (black lanes separate blots that were derived from the same gel, but were noncontiguous). ( B ) IFN-α–induced OAS1 mRNA expression relative to GAPDH was assessed by qRT-PCR. Cells were treated with 1,000 U/ml IFN-α for 8 hours. Upregulation of OAS1 was found only in cells with WT STAT1 after IFN-α treatment. Expression of maximal amounts of Y701F-mutated STAT1 in U3A cells did not induce ISG expression. Shown are the mean values with SEM of three replicate experiments.

Techniques Used: Clone Assay, Expressing, Derivative Assay, Quantitative RT-PCR

pegIFN-α2b transiently induces the Jak/STAT pathway in the liver. ( A ) Representative images of IHC analysis of p-STAT1 in liver biopsies obtained before treatment (B1) and at several time points after the first injection of pegIFN-α2b. Strong nuclear p-STAT1 signals were present at the 4- and 16-hour time points, but not at later time points, where the signals were localized in nonparenchymal cells (arrows). Scale bars: 20 μm. ( B ) Quantitative analysis of the mean percentage of p-STAT1–positive hepatocyte nuclei (5 × 100 cells counted per sample; the number of samples is indicated) per time point. Bars show the mean with SEM.
Figure Legend Snippet: pegIFN-α2b transiently induces the Jak/STAT pathway in the liver. ( A ) Representative images of IHC analysis of p-STAT1 in liver biopsies obtained before treatment (B1) and at several time points after the first injection of pegIFN-α2b. Strong nuclear p-STAT1 signals were present at the 4- and 16-hour time points, but not at later time points, where the signals were localized in nonparenchymal cells (arrows). Scale bars: 20 μm. ( B ) Quantitative analysis of the mean percentage of p-STAT1–positive hepatocyte nuclei (5 × 100 cells counted per sample; the number of samples is indicated) per time point. Bars show the mean with SEM.

Techniques Used: Immunohistochemistry, Injection

11) Product Images from "Structurally Modified Curcumin Analogs Inhibit STAT3 Phosphorylation and Promote Apoptosis of Human Renal Cell Carcinoma and Melanoma Cell Lines"

Article Title: Structurally Modified Curcumin Analogs Inhibit STAT3 Phosphorylation and Promote Apoptosis of Human Renal Cell Carcinoma and Melanoma Cell Lines

Journal: PLoS ONE

doi: 10.1371/journal.pone.0040724

IFN-γ-induced signal transduction, in the presence of FLLL32 and FLLL62. (A) Human ACHN RCC cells or (B) A375 melanoma cells were pre-treated for 16 hours with DMSO (negative control), multiple doses of FLLL32, FLLL62, or curcumin and subsequently stimulated with IFN-γ (10 ng/mL) or PBS (vehicle) for 15 minutes. The expression of pSTAT1 and pSTAT3 were evaluated by immunoblot. Membranes were probed for total STAT3 protein, total STAT1 protein and β-actin to control for loading.
Figure Legend Snippet: IFN-γ-induced signal transduction, in the presence of FLLL32 and FLLL62. (A) Human ACHN RCC cells or (B) A375 melanoma cells were pre-treated for 16 hours with DMSO (negative control), multiple doses of FLLL32, FLLL62, or curcumin and subsequently stimulated with IFN-γ (10 ng/mL) or PBS (vehicle) for 15 minutes. The expression of pSTAT1 and pSTAT3 were evaluated by immunoblot. Membranes were probed for total STAT3 protein, total STAT1 protein and β-actin to control for loading.

Techniques Used: Transduction, Negative Control, Expressing

12) Product Images from "The STAT4 SLE risk allele rs7574865[T] is associated with increased IL-12-induced IFN-γ production in T cells from patients with SLE"

Article Title: The STAT4 SLE risk allele rs7574865[T] is associated with increased IL-12-induced IFN-γ production in T cells from patients with SLE

Journal: Annals of the Rheumatic Diseases

doi: 10.1136/annrheumdis-2017-212794

Janus kinase inhibitors block the IL-12 and IFN-γ response in peripheral blood mononuclear cells (PBMCs) from STAT4 risk patients with systemic lupus erythematosus (SLE). (A) Healthy donor PBMCs were treated with serial dilutions of the JAK2 selective (JAK2i), the TYK2 selective (TYK2i) and the pan-JAK inhibitor (pan-JAKi) before being stimulated with 5 ng/mL IL-12, 0.1 ng/mL IFN-γ or 200 U/mL IFN-α for 20 min. IL-12-stimulated cells were preactivated with PHA/IL-2. Phosphorylation of STAT4 (pSTAT4) and STAT1 (pSTAT1) was determined by flow cytometry in IL-12-stimulated cells and IFN-γ or IFN-α-stimulated cells, respectively. Data (mean±SD of two individuals) from IL-12 and IFN-α-stimulated cells are shown for CD3 + T cells and from IFN-γ-stimulated cells for monocytes. (B) IC 50 values for each inhibitor. (C–E) PBMCs from healthy donors (HD), patients with SLE homozygous for the protective STAT4 allele (G/G) and patients with SLE carrying one or two STAT4 risk alleles (G/T+T/T) were incubated with 200 nM TYK2i (C, D), 70 nm JAK2i or 30 nM pan-JAKi (E). (C, D) Inhibition of IL-12-induced pSTAT4 in CD8 + T cells (C) and reduction in the frequency of IFN-γ + memory CD45RO + CD57 – CD8 + T cells (D). (E) Inhibition of IFN-γ-induced pSTAT1 in monocytes. (C–E) Open triangles and squares denote G/T and T/T patients with SLE, respectively.
Figure Legend Snippet: Janus kinase inhibitors block the IL-12 and IFN-γ response in peripheral blood mononuclear cells (PBMCs) from STAT4 risk patients with systemic lupus erythematosus (SLE). (A) Healthy donor PBMCs were treated with serial dilutions of the JAK2 selective (JAK2i), the TYK2 selective (TYK2i) and the pan-JAK inhibitor (pan-JAKi) before being stimulated with 5 ng/mL IL-12, 0.1 ng/mL IFN-γ or 200 U/mL IFN-α for 20 min. IL-12-stimulated cells were preactivated with PHA/IL-2. Phosphorylation of STAT4 (pSTAT4) and STAT1 (pSTAT1) was determined by flow cytometry in IL-12-stimulated cells and IFN-γ or IFN-α-stimulated cells, respectively. Data (mean±SD of two individuals) from IL-12 and IFN-α-stimulated cells are shown for CD3 + T cells and from IFN-γ-stimulated cells for monocytes. (B) IC 50 values for each inhibitor. (C–E) PBMCs from healthy donors (HD), patients with SLE homozygous for the protective STAT4 allele (G/G) and patients with SLE carrying one or two STAT4 risk alleles (G/T+T/T) were incubated with 200 nM TYK2i (C, D), 70 nm JAK2i or 30 nM pan-JAKi (E). (C, D) Inhibition of IL-12-induced pSTAT4 in CD8 + T cells (C) and reduction in the frequency of IFN-γ + memory CD45RO + CD57 – CD8 + T cells (D). (E) Inhibition of IFN-γ-induced pSTAT1 in monocytes. (C–E) Open triangles and squares denote G/T and T/T patients with SLE, respectively.

Techniques Used: Blocking Assay, Flow Cytometry, Cytometry, Incubation, Inhibition

The STAT4 risk allele does not affect IFN-α, IFN-γ or IL-12-induced phosphorylation of STAT4 or STAT1 in resting peripheral blood mononuclear cells (PBMCs). PBMCs from patients with systemic lupus erythematosus (SLE) were stimulated with 2000 U/mL IFN-α (A–C), 1 ng/mL IFN-γ (A, D) or 10 ng/mL IL-12 (A, E) for 20 min and phosphorylation of STAT4 (pSTAT4) and STAT1 (pSTAT1) was determined in indicated cell population, by flow cytometry. (A) Flow cytometry plots with gating strategy from one representative donor. (B–E) Cumulative data from 19 homozygous protective (G/G, black circles), 21 heterozygous (G/T, open triangles) and 9 homozygous risk (T/T, open squares) STAT4 patients with SLE. For monocytes, frequencies of pSTAT4 + and pSTAT1 + cells are shown because of the bimodal distributions. Horizontal red bars indicate the mean value. Due to
Figure Legend Snippet: The STAT4 risk allele does not affect IFN-α, IFN-γ or IL-12-induced phosphorylation of STAT4 or STAT1 in resting peripheral blood mononuclear cells (PBMCs). PBMCs from patients with systemic lupus erythematosus (SLE) were stimulated with 2000 U/mL IFN-α (A–C), 1 ng/mL IFN-γ (A, D) or 10 ng/mL IL-12 (A, E) for 20 min and phosphorylation of STAT4 (pSTAT4) and STAT1 (pSTAT1) was determined in indicated cell population, by flow cytometry. (A) Flow cytometry plots with gating strategy from one representative donor. (B–E) Cumulative data from 19 homozygous protective (G/G, black circles), 21 heterozygous (G/T, open triangles) and 9 homozygous risk (T/T, open squares) STAT4 patients with SLE. For monocytes, frequencies of pSTAT4 + and pSTAT1 + cells are shown because of the bimodal distributions. Horizontal red bars indicate the mean value. Due to

Techniques Used: Flow Cytometry, Cytometry

13) Product Images from "Human metapneumovirus small hydrophobic (SH) protein downregulates type I IFN pathway signaling by affecting STAT1 expression and phosphorylation"

Article Title: Human metapneumovirus small hydrophobic (SH) protein downregulates type I IFN pathway signaling by affecting STAT1 expression and phosphorylation

Journal: Virology

doi: 10.1016/j.virol.2016.04.022

HMPV inhibits STAT1 phosphorylation of Vero cells only in infected cells. (A) Vero cells were infected for 24 h with HMPV at MOI of 1 in 6-well plates. Cells were lysed with RIPA buffer, run on 4-15% bis-tris gel, transferred to nitrocellulose membrane,
Figure Legend Snippet: HMPV inhibits STAT1 phosphorylation of Vero cells only in infected cells. (A) Vero cells were infected for 24 h with HMPV at MOI of 1 in 6-well plates. Cells were lysed with RIPA buffer, run on 4-15% bis-tris gel, transferred to nitrocellulose membrane,

Techniques Used: Infection

Type I IFN secreted by infected BEAS-2B cells inhibits STAT1 phosphorylation in neighboring uninfected cells. (A) BEAS-2B cells were infected with HMPV for 24 h at an MOI of 1 in 6-well plates, then stained with polyclonal anti-HMPV antibody and gated
Figure Legend Snippet: Type I IFN secreted by infected BEAS-2B cells inhibits STAT1 phosphorylation in neighboring uninfected cells. (A) BEAS-2B cells were infected with HMPV for 24 h at an MOI of 1 in 6-well plates, then stained with polyclonal anti-HMPV antibody and gated

Techniques Used: Infection, Staining

HMPV inhibits STAT1 phosphorylation in BEAS-2B cells. (A) BEAS-2B cells were infected for 24 h with HMPV at an MOI of 1 in 6-well plates. Cells were lysed with RIPA buffer, run on 4-15% bis-tris gel, transferred to nitrocellulose membrane, and probed
Figure Legend Snippet: HMPV inhibits STAT1 phosphorylation in BEAS-2B cells. (A) BEAS-2B cells were infected for 24 h with HMPV at an MOI of 1 in 6-well plates. Cells were lysed with RIPA buffer, run on 4-15% bis-tris gel, transferred to nitrocellulose membrane, and probed

Techniques Used: Infection

Transient expression of HMPV SH protein inhibits STAT1 phosphorylation. (A) Vero cells transfected with HMPV GFP-fusion proteins were analyzed compared to untransfected cells in the same culture using flow cytometry. (B) Transfected cells were analyzed
Figure Legend Snippet: Transient expression of HMPV SH protein inhibits STAT1 phosphorylation. (A) Vero cells transfected with HMPV GFP-fusion proteins were analyzed compared to untransfected cells in the same culture using flow cytometry. (B) Transfected cells were analyzed

Techniques Used: Expressing, Transfection, Flow Cytometry, Cytometry

14) Product Images from "Human metapneumovirus small hydrophobic (SH) protein downregulates type I IFN pathway signaling by affecting STAT1 expression and phosphorylation"

Article Title: Human metapneumovirus small hydrophobic (SH) protein downregulates type I IFN pathway signaling by affecting STAT1 expression and phosphorylation

Journal: Virology

doi: 10.1016/j.virol.2016.04.022

HMPV inhibits STAT1 phosphorylation of Vero cells only in infected cells. (A) Vero cells were infected for 24 h with HMPV at MOI of 1 in 6-well plates. Cells were lysed with RIPA buffer, run on 4-15% bis-tris gel, transferred to nitrocellulose membrane,
Figure Legend Snippet: HMPV inhibits STAT1 phosphorylation of Vero cells only in infected cells. (A) Vero cells were infected for 24 h with HMPV at MOI of 1 in 6-well plates. Cells were lysed with RIPA buffer, run on 4-15% bis-tris gel, transferred to nitrocellulose membrane,

Techniques Used: Infection

Type I IFN secreted by infected BEAS-2B cells inhibits STAT1 phosphorylation in neighboring uninfected cells. (A) BEAS-2B cells were infected with HMPV for 24 h at an MOI of 1 in 6-well plates, then stained with polyclonal anti-HMPV antibody and gated
Figure Legend Snippet: Type I IFN secreted by infected BEAS-2B cells inhibits STAT1 phosphorylation in neighboring uninfected cells. (A) BEAS-2B cells were infected with HMPV for 24 h at an MOI of 1 in 6-well plates, then stained with polyclonal anti-HMPV antibody and gated

Techniques Used: Infection, Staining

HMPV inhibits STAT1 phosphorylation in BEAS-2B cells. (A) BEAS-2B cells were infected for 24 h with HMPV at an MOI of 1 in 6-well plates. Cells were lysed with RIPA buffer, run on 4-15% bis-tris gel, transferred to nitrocellulose membrane, and probed
Figure Legend Snippet: HMPV inhibits STAT1 phosphorylation in BEAS-2B cells. (A) BEAS-2B cells were infected for 24 h with HMPV at an MOI of 1 in 6-well plates. Cells were lysed with RIPA buffer, run on 4-15% bis-tris gel, transferred to nitrocellulose membrane, and probed

Techniques Used: Infection

Transient expression of HMPV SH protein inhibits STAT1 phosphorylation. (A) Vero cells transfected with HMPV GFP-fusion proteins were analyzed compared to untransfected cells in the same culture using flow cytometry. (B) Transfected cells were analyzed
Figure Legend Snippet: Transient expression of HMPV SH protein inhibits STAT1 phosphorylation. (A) Vero cells transfected with HMPV GFP-fusion proteins were analyzed compared to untransfected cells in the same culture using flow cytometry. (B) Transfected cells were analyzed

Techniques Used: Expressing, Transfection, Flow Cytometry, Cytometry

15) Product Images from "Human metapneumovirus small hydrophobic (SH) protein downregulates type I IFN pathway signaling by affecting STAT1 expression and phosphorylation"

Article Title: Human metapneumovirus small hydrophobic (SH) protein downregulates type I IFN pathway signaling by affecting STAT1 expression and phosphorylation

Journal: Virology

doi: 10.1016/j.virol.2016.04.022

HMPV inhibits STAT1 phosphorylation of Vero cells only in infected cells. (A) Vero cells were infected for 24 h with HMPV at MOI of 1 in 6-well plates. Cells were lysed with RIPA buffer, run on 4-15% bis-tris gel, transferred to nitrocellulose membrane,
Figure Legend Snippet: HMPV inhibits STAT1 phosphorylation of Vero cells only in infected cells. (A) Vero cells were infected for 24 h with HMPV at MOI of 1 in 6-well plates. Cells were lysed with RIPA buffer, run on 4-15% bis-tris gel, transferred to nitrocellulose membrane,

Techniques Used: Infection

Type I IFN secreted by infected BEAS-2B cells inhibits STAT1 phosphorylation in neighboring uninfected cells. (A) BEAS-2B cells were infected with HMPV for 24 h at an MOI of 1 in 6-well plates, then stained with polyclonal anti-HMPV antibody and gated
Figure Legend Snippet: Type I IFN secreted by infected BEAS-2B cells inhibits STAT1 phosphorylation in neighboring uninfected cells. (A) BEAS-2B cells were infected with HMPV for 24 h at an MOI of 1 in 6-well plates, then stained with polyclonal anti-HMPV antibody and gated

Techniques Used: Infection, Staining

HMPV inhibits STAT1 phosphorylation in BEAS-2B cells. (A) BEAS-2B cells were infected for 24 h with HMPV at an MOI of 1 in 6-well plates. Cells were lysed with RIPA buffer, run on 4-15% bis-tris gel, transferred to nitrocellulose membrane, and probed
Figure Legend Snippet: HMPV inhibits STAT1 phosphorylation in BEAS-2B cells. (A) BEAS-2B cells were infected for 24 h with HMPV at an MOI of 1 in 6-well plates. Cells were lysed with RIPA buffer, run on 4-15% bis-tris gel, transferred to nitrocellulose membrane, and probed

Techniques Used: Infection

Transient expression of HMPV SH protein inhibits STAT1 phosphorylation. (A) Vero cells transfected with HMPV GFP-fusion proteins were analyzed compared to untransfected cells in the same culture using flow cytometry. (B) Transfected cells were analyzed
Figure Legend Snippet: Transient expression of HMPV SH protein inhibits STAT1 phosphorylation. (A) Vero cells transfected with HMPV GFP-fusion proteins were analyzed compared to untransfected cells in the same culture using flow cytometry. (B) Transfected cells were analyzed

Techniques Used: Expressing, Transfection, Flow Cytometry, Cytometry

16) Product Images from "Human metapneumovirus small hydrophobic (SH) protein downregulates type I IFN pathway signaling by affecting STAT1 expression and phosphorylation"

Article Title: Human metapneumovirus small hydrophobic (SH) protein downregulates type I IFN pathway signaling by affecting STAT1 expression and phosphorylation

Journal: Virology

doi: 10.1016/j.virol.2016.04.022

HMPV inhibits STAT1 phosphorylation of Vero cells only in infected cells. (A) Vero cells were infected for 24 h with HMPV at MOI of 1 in 6-well plates. Cells were lysed with RIPA buffer, run on 4-15% bis-tris gel, transferred to nitrocellulose membrane,
Figure Legend Snippet: HMPV inhibits STAT1 phosphorylation of Vero cells only in infected cells. (A) Vero cells were infected for 24 h with HMPV at MOI of 1 in 6-well plates. Cells were lysed with RIPA buffer, run on 4-15% bis-tris gel, transferred to nitrocellulose membrane,

Techniques Used: Infection

Type I IFN secreted by infected BEAS-2B cells inhibits STAT1 phosphorylation in neighboring uninfected cells. (A) BEAS-2B cells were infected with HMPV for 24 h at an MOI of 1 in 6-well plates, then stained with polyclonal anti-HMPV antibody and gated
Figure Legend Snippet: Type I IFN secreted by infected BEAS-2B cells inhibits STAT1 phosphorylation in neighboring uninfected cells. (A) BEAS-2B cells were infected with HMPV for 24 h at an MOI of 1 in 6-well plates, then stained with polyclonal anti-HMPV antibody and gated

Techniques Used: Infection, Staining

HMPV inhibits STAT1 phosphorylation in BEAS-2B cells. (A) BEAS-2B cells were infected for 24 h with HMPV at an MOI of 1 in 6-well plates. Cells were lysed with RIPA buffer, run on 4-15% bis-tris gel, transferred to nitrocellulose membrane, and probed
Figure Legend Snippet: HMPV inhibits STAT1 phosphorylation in BEAS-2B cells. (A) BEAS-2B cells were infected for 24 h with HMPV at an MOI of 1 in 6-well plates. Cells were lysed with RIPA buffer, run on 4-15% bis-tris gel, transferred to nitrocellulose membrane, and probed

Techniques Used: Infection

Transient expression of HMPV SH protein inhibits STAT1 phosphorylation. (A) Vero cells transfected with HMPV GFP-fusion proteins were analyzed compared to untransfected cells in the same culture using flow cytometry. (B) Transfected cells were analyzed
Figure Legend Snippet: Transient expression of HMPV SH protein inhibits STAT1 phosphorylation. (A) Vero cells transfected with HMPV GFP-fusion proteins were analyzed compared to untransfected cells in the same culture using flow cytometry. (B) Transfected cells were analyzed

Techniques Used: Expressing, Transfection, Flow Cytometry, Cytometry

17) Product Images from "NS5 of Dengue Virus Mediates STAT2 Binding and Degradation ▿"

Article Title: NS5 of Dengue Virus Mediates STAT2 Binding and Degradation ▿

Journal: Journal of Virology

doi: 10.1128/JVI.02188-08

Truncations in the DENV NS5 protein affect its ability to associate with STAT2, decrease STAT2 levels, and inhibit IFN signaling. (A) 293T cells were transfected with the indicated constructs. Numbering refers to the glycine start position within the NS5 protein. All numbered forms of NS5 are expressed within the context of the E-Ub-NS5-GFP construct (i.e., 10-900, NS5 residues 10 to 900 fused to the C terminus of the E-Ub cassette). Twenty-four hours posttransfection, cells were sorted for GFP-positive cells using FACS, lysed, and examined by Western blotting using STAT2, GFP, and tubulin antibodies. (B) 293T cells were cotransfected with the indicated plasmids (the numbering system is identical to that described in A), STAT1-FLAG, and STAT2-FLAG. In order to detect STAT2 binding by NS5, STAT2-FLAG plasmid was transfected in excess with respect to E-Ub-NS5-GFP plasmids, resulting in a not-detectable degradation of STAT2-FLAG. Lysates were immunoprecipitated with a polyclonal GFP antibody (pGFP), and Western blots were performed using FLAG, GFP, and GAPDH antibodies. TCE, total cell extracts. (C) 293T cells were transfected with the ISRE-54-CAT reporter, a constitutively expressing firefly luciferase plasmid, and the indicated HA-tagged viral protein. Twenty-four hours posttransfection, cells were treated with 1,000 U/ml of type I IFN. Twenty-four hours posttreatment, cells were lysed and measured for CAT and luciferase activity. Data are represented with the standard deviations from three independent experiments. Samples showing P values of less than 0.05 compared with the empty control sample are indicated.
Figure Legend Snippet: Truncations in the DENV NS5 protein affect its ability to associate with STAT2, decrease STAT2 levels, and inhibit IFN signaling. (A) 293T cells were transfected with the indicated constructs. Numbering refers to the glycine start position within the NS5 protein. All numbered forms of NS5 are expressed within the context of the E-Ub-NS5-GFP construct (i.e., 10-900, NS5 residues 10 to 900 fused to the C terminus of the E-Ub cassette). Twenty-four hours posttransfection, cells were sorted for GFP-positive cells using FACS, lysed, and examined by Western blotting using STAT2, GFP, and tubulin antibodies. (B) 293T cells were cotransfected with the indicated plasmids (the numbering system is identical to that described in A), STAT1-FLAG, and STAT2-FLAG. In order to detect STAT2 binding by NS5, STAT2-FLAG plasmid was transfected in excess with respect to E-Ub-NS5-GFP plasmids, resulting in a not-detectable degradation of STAT2-FLAG. Lysates were immunoprecipitated with a polyclonal GFP antibody (pGFP), and Western blots were performed using FLAG, GFP, and GAPDH antibodies. TCE, total cell extracts. (C) 293T cells were transfected with the ISRE-54-CAT reporter, a constitutively expressing firefly luciferase plasmid, and the indicated HA-tagged viral protein. Twenty-four hours posttransfection, cells were treated with 1,000 U/ml of type I IFN. Twenty-four hours posttreatment, cells were lysed and measured for CAT and luciferase activity. Data are represented with the standard deviations from three independent experiments. Samples showing P values of less than 0.05 compared with the empty control sample are indicated.

Techniques Used: Transfection, Construct, FACS, Western Blot, Binding Assay, Plasmid Preparation, Immunoprecipitation, Expressing, Luciferase, Activity Assay

Inhibitors of the ubiquitin-proteasome pathway prevent STAT2 degradation by DENV NS5. (A) wtVero or Vero cells stably expressing the DEN1 replicon were treated with the indicated amounts of MG132. Sixteen hours posttreatment, cells were lysed and examined for ubiquitin, STAT2, STAT1, NS5, and GAPDH levels via Western blotting. (B) STAT2-deficient U6A cells were transfected with HA-ubiquitin, STAT2-FLAG, STAT1-GFP, NS2b-3, and either E- clv NS5-HA or an empty vector plasmid. Cells were then treated with lactacystin for 8 h and subsequently lysed and examined by Western blotting using ubiquitin, STAT2, STAT1, HA, and tubulin antibodies. (C) 293T cells were cotransfected with NS2b-3-HA and the plasmids indicated at the top. Ten hours posttransfection, cells were treated with the indicated amounts of lactacystin. Twenty-four hours posttransfection, cells were sorted for GFP-positive cells by FACS, lysed, and examined by Western blotting using ubiquitin-, GFP-, HA-, and GAPDH-specific antibodies. (D) STAT2-deficient U6A cells were transfected with 0.1 or 1 μg of FLAG-STAT2-FLAG, 0.1 μg NS2b-3, and either 1 μg E- clv NS5-HA or an empty vector plasmid. Cells were then lysed and examined by Western blotting using FLAG antibody and long-term film exposure to detect any additional low-intensity bands. The arrow indicates the expected size of FLAG-STAT2-FLAG. The asterisk marks a nonspecific band running at the same mobility of FLAG-STAT2-FLAG. (E) 293T cells were cotransfected with NS2b-3 and the plasmids indicated at the top. Twenty-four hours posttransfection, cells were sorted for GFP-positive cells by FACS, lysed, and examined via Western blotting using STAT2-, GFP-, HA-, FLAG-, and GAPDH-specific antibodies.
Figure Legend Snippet: Inhibitors of the ubiquitin-proteasome pathway prevent STAT2 degradation by DENV NS5. (A) wtVero or Vero cells stably expressing the DEN1 replicon were treated with the indicated amounts of MG132. Sixteen hours posttreatment, cells were lysed and examined for ubiquitin, STAT2, STAT1, NS5, and GAPDH levels via Western blotting. (B) STAT2-deficient U6A cells were transfected with HA-ubiquitin, STAT2-FLAG, STAT1-GFP, NS2b-3, and either E- clv NS5-HA or an empty vector plasmid. Cells were then treated with lactacystin for 8 h and subsequently lysed and examined by Western blotting using ubiquitin, STAT2, STAT1, HA, and tubulin antibodies. (C) 293T cells were cotransfected with NS2b-3-HA and the plasmids indicated at the top. Ten hours posttransfection, cells were treated with the indicated amounts of lactacystin. Twenty-four hours posttransfection, cells were sorted for GFP-positive cells by FACS, lysed, and examined by Western blotting using ubiquitin-, GFP-, HA-, and GAPDH-specific antibodies. (D) STAT2-deficient U6A cells were transfected with 0.1 or 1 μg of FLAG-STAT2-FLAG, 0.1 μg NS2b-3, and either 1 μg E- clv NS5-HA or an empty vector plasmid. Cells were then lysed and examined by Western blotting using FLAG antibody and long-term film exposure to detect any additional low-intensity bands. The arrow indicates the expected size of FLAG-STAT2-FLAG. The asterisk marks a nonspecific band running at the same mobility of FLAG-STAT2-FLAG. (E) 293T cells were cotransfected with NS2b-3 and the plasmids indicated at the top. Twenty-four hours posttransfection, cells were sorted for GFP-positive cells by FACS, lysed, and examined via Western blotting using STAT2-, GFP-, HA-, FLAG-, and GAPDH-specific antibodies.

Techniques Used: Stable Transfection, Expressing, Western Blot, Transfection, Plasmid Preparation, FACS

DENV NS5 interacts with STAT2. (A) 293T cells were cotransfected with plasmids expressing FLAG-tagged STAT1 (STAT1-FLAG) and STAT2 (STAT2-FLAG) and empty plasmid (empty) or plasmid expressing HA-tagged DENV NS5 (NS5-HA), DENV core (CORE-HA), or NiV-V (HA-NiV-V) proteins. Lysates were then immunoprecipitated with anti-HA antibody (IP HA), and Western blotting was performed using anti-HA and anti-FLAG antibodies. Asterisks mark the heavy and light chains from the HA antibody. (B) 293T cells were cotransfected with plasmids expressing NS5-HA and either STAT1-FLAG or STAT2-FLAG. Lysates were then immunoprecipitated with anti-FLAG antibody (IP FLAG), and Western blotting was performed using anti-HA and anti-FLAG antibodies. TCE, total cell extracts were subjected to Western blotting using anti-HA, anti-FLAG, and anti-GAPDH antibodies.
Figure Legend Snippet: DENV NS5 interacts with STAT2. (A) 293T cells were cotransfected with plasmids expressing FLAG-tagged STAT1 (STAT1-FLAG) and STAT2 (STAT2-FLAG) and empty plasmid (empty) or plasmid expressing HA-tagged DENV NS5 (NS5-HA), DENV core (CORE-HA), or NiV-V (HA-NiV-V) proteins. Lysates were then immunoprecipitated with anti-HA antibody (IP HA), and Western blotting was performed using anti-HA and anti-FLAG antibodies. Asterisks mark the heavy and light chains from the HA antibody. (B) 293T cells were cotransfected with plasmids expressing NS5-HA and either STAT1-FLAG or STAT2-FLAG. Lysates were then immunoprecipitated with anti-FLAG antibody (IP FLAG), and Western blotting was performed using anti-HA and anti-FLAG antibodies. TCE, total cell extracts were subjected to Western blotting using anti-HA, anti-FLAG, and anti-GAPDH antibodies.

Techniques Used: Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot

Expression of a precursor form of DENV NS5 cleaved by the DENV protease results in reduced STAT2 levels. (A) 293T cells were transfected with the indicated constructs. Twenty-four hours posttransfection, cells were sorted for GFP-positive cells by FACS, subsequently lysed, and examined via Western blotting using GFP-, HA-, STAT1-, STAT2-, and GAPDH-specific antibodies. Schematics of the transfected constructs are shown at the bottom. ORFs that contain an “sp” at the N terminus have a signal peptide which directs the entire polyprotein to the surface of the ER for translation. Black arrows and red arrows indicate cleavage sites for cellular and the DEN2 viral proteases, respectively. The DEN2 active protease is highlighted in red. Densitometry analysis of the levels of STAT2 and NS5 are included on the far right, and levels are calculated relative to the levels in lane 1, with a value of 1 in the case of NS5 levels being indicative of no detection (background levels). (B) Same as above (A). Mutation of the DENV protease recognition site at the N terminus of NS5 is indicated by the blue dashes. The DENV protease labeled in blue indicates a serine-to-alanine mutation within the catalytic site of the DENV protease.
Figure Legend Snippet: Expression of a precursor form of DENV NS5 cleaved by the DENV protease results in reduced STAT2 levels. (A) 293T cells were transfected with the indicated constructs. Twenty-four hours posttransfection, cells were sorted for GFP-positive cells by FACS, subsequently lysed, and examined via Western blotting using GFP-, HA-, STAT1-, STAT2-, and GAPDH-specific antibodies. Schematics of the transfected constructs are shown at the bottom. ORFs that contain an “sp” at the N terminus have a signal peptide which directs the entire polyprotein to the surface of the ER for translation. Black arrows and red arrows indicate cleavage sites for cellular and the DEN2 viral proteases, respectively. The DEN2 active protease is highlighted in red. Densitometry analysis of the levels of STAT2 and NS5 are included on the far right, and levels are calculated relative to the levels in lane 1, with a value of 1 in the case of NS5 levels being indicative of no detection (background levels). (B) Same as above (A). Mutation of the DENV protease recognition site at the N terminus of NS5 is indicated by the blue dashes. The DENV protease labeled in blue indicates a serine-to-alanine mutation within the catalytic site of the DENV protease.

Techniques Used: Expressing, Transfection, Construct, FACS, Western Blot, Mutagenesis, Labeling

Cleaved NS5 is sufficient for reduced STAT2 levels and does not require an N-terminal glycine. (A) 293T cells were transfected with the indicated constructs. Twenty-four hours posttransfection, cells were sorted for GFP-positive cells by FACS, subsequently lysed, subjected to 4-to-20% SDS-polyacrylamide gel electrophoresis, and examined via Western blotting using GFP-, HA-, STAT1-, STAT2-, and GAPDH-specific antibodies. The TEV protease and its cleavage site are indicated by the green text and arrow, respectively. Cleavage sequences targeted by endogenous deubiquitinases are noted by a yellow arrow. Densitometry analyses of the levels of STAT2 and NS5 are included at the bottom, and levels were calculated relative to the levels in lane 1, with a value of 1 in the case of NS5 levels being indicative of no detection (background levels). (B) Same as above (A) except that lysates were run on a 7.5% SDS-polyacrylamide gel and subsequently analyzed by Western blotting using GFP-, STAT2-, and tubulin-specific antibodies.
Figure Legend Snippet: Cleaved NS5 is sufficient for reduced STAT2 levels and does not require an N-terminal glycine. (A) 293T cells were transfected with the indicated constructs. Twenty-four hours posttransfection, cells were sorted for GFP-positive cells by FACS, subsequently lysed, subjected to 4-to-20% SDS-polyacrylamide gel electrophoresis, and examined via Western blotting using GFP-, HA-, STAT1-, STAT2-, and GAPDH-specific antibodies. The TEV protease and its cleavage site are indicated by the green text and arrow, respectively. Cleavage sequences targeted by endogenous deubiquitinases are noted by a yellow arrow. Densitometry analyses of the levels of STAT2 and NS5 are included at the bottom, and levels were calculated relative to the levels in lane 1, with a value of 1 in the case of NS5 levels being indicative of no detection (background levels). (B) Same as above (A) except that lysates were run on a 7.5% SDS-polyacrylamide gel and subsequently analyzed by Western blotting using GFP-, STAT2-, and tubulin-specific antibodies.

Techniques Used: Transfection, Construct, FACS, Polyacrylamide Gel Electrophoresis, Western Blot

18) Product Images from "NS5 of Dengue Virus Mediates STAT2 Binding and Degradation ▿"

Article Title: NS5 of Dengue Virus Mediates STAT2 Binding and Degradation ▿

Journal: Journal of Virology

doi: 10.1128/JVI.02188-08

Truncations in the DENV NS5 protein affect its ability to associate with STAT2, decrease STAT2 levels, and inhibit IFN signaling. (A) 293T cells were transfected with the indicated constructs. Numbering refers to the glycine start position within the NS5 protein. All numbered forms of NS5 are expressed within the context of the E-Ub-NS5-GFP construct (i.e., 10-900, NS5 residues 10 to 900 fused to the C terminus of the E-Ub cassette). Twenty-four hours posttransfection, cells were sorted for GFP-positive cells using FACS, lysed, and examined by Western blotting using STAT2, GFP, and tubulin antibodies. (B) 293T cells were cotransfected with the indicated plasmids (the numbering system is identical to that described in A), STAT1-FLAG, and STAT2-FLAG. In order to detect STAT2 binding by NS5, STAT2-FLAG plasmid was transfected in excess with respect to E-Ub-NS5-GFP plasmids, resulting in a not-detectable degradation of STAT2-FLAG. Lysates were immunoprecipitated with a polyclonal GFP antibody (pGFP), and Western blots were performed using FLAG, GFP, and GAPDH antibodies. TCE, total cell extracts. (C) 293T cells were transfected with the ISRE-54-CAT reporter, a constitutively expressing firefly luciferase plasmid, and the indicated HA-tagged viral protein. Twenty-four hours posttransfection, cells were treated with 1,000 U/ml of type I IFN. Twenty-four hours posttreatment, cells were lysed and measured for CAT and luciferase activity. Data are represented with the standard deviations from three independent experiments. Samples showing P values of less than 0.05 compared with the empty control sample are indicated.
Figure Legend Snippet: Truncations in the DENV NS5 protein affect its ability to associate with STAT2, decrease STAT2 levels, and inhibit IFN signaling. (A) 293T cells were transfected with the indicated constructs. Numbering refers to the glycine start position within the NS5 protein. All numbered forms of NS5 are expressed within the context of the E-Ub-NS5-GFP construct (i.e., 10-900, NS5 residues 10 to 900 fused to the C terminus of the E-Ub cassette). Twenty-four hours posttransfection, cells were sorted for GFP-positive cells using FACS, lysed, and examined by Western blotting using STAT2, GFP, and tubulin antibodies. (B) 293T cells were cotransfected with the indicated plasmids (the numbering system is identical to that described in A), STAT1-FLAG, and STAT2-FLAG. In order to detect STAT2 binding by NS5, STAT2-FLAG plasmid was transfected in excess with respect to E-Ub-NS5-GFP plasmids, resulting in a not-detectable degradation of STAT2-FLAG. Lysates were immunoprecipitated with a polyclonal GFP antibody (pGFP), and Western blots were performed using FLAG, GFP, and GAPDH antibodies. TCE, total cell extracts. (C) 293T cells were transfected with the ISRE-54-CAT reporter, a constitutively expressing firefly luciferase plasmid, and the indicated HA-tagged viral protein. Twenty-four hours posttransfection, cells were treated with 1,000 U/ml of type I IFN. Twenty-four hours posttreatment, cells were lysed and measured for CAT and luciferase activity. Data are represented with the standard deviations from three independent experiments. Samples showing P values of less than 0.05 compared with the empty control sample are indicated.

Techniques Used: Transfection, Construct, FACS, Western Blot, Binding Assay, Plasmid Preparation, Immunoprecipitation, Expressing, Luciferase, Activity Assay

Inhibitors of the ubiquitin-proteasome pathway prevent STAT2 degradation by DENV NS5. (A) wtVero or Vero cells stably expressing the DEN1 replicon were treated with the indicated amounts of MG132. Sixteen hours posttreatment, cells were lysed and examined for ubiquitin, STAT2, STAT1, NS5, and GAPDH levels via Western blotting. (B) STAT2-deficient U6A cells were transfected with HA-ubiquitin, STAT2-FLAG, STAT1-GFP, NS2b-3, and either E- clv NS5-HA or an empty vector plasmid. Cells were then treated with lactacystin for 8 h and subsequently lysed and examined by Western blotting using ubiquitin, STAT2, STAT1, HA, and tubulin antibodies. (C) 293T cells were cotransfected with NS2b-3-HA and the plasmids indicated at the top. Ten hours posttransfection, cells were treated with the indicated amounts of lactacystin. Twenty-four hours posttransfection, cells were sorted for GFP-positive cells by FACS, lysed, and examined by Western blotting using ubiquitin-, GFP-, HA-, and GAPDH-specific antibodies. (D) STAT2-deficient U6A cells were transfected with 0.1 or 1 μg of FLAG-STAT2-FLAG, 0.1 μg NS2b-3, and either 1 μg E- clv NS5-HA or an empty vector plasmid. Cells were then lysed and examined by Western blotting using FLAG antibody and long-term film exposure to detect any additional low-intensity bands. The arrow indicates the expected size of FLAG-STAT2-FLAG. The asterisk marks a nonspecific band running at the same mobility of FLAG-STAT2-FLAG. (E) 293T cells were cotransfected with NS2b-3 and the plasmids indicated at the top. Twenty-four hours posttransfection, cells were sorted for GFP-positive cells by FACS, lysed, and examined via Western blotting using STAT2-, GFP-, HA-, FLAG-, and GAPDH-specific antibodies.
Figure Legend Snippet: Inhibitors of the ubiquitin-proteasome pathway prevent STAT2 degradation by DENV NS5. (A) wtVero or Vero cells stably expressing the DEN1 replicon were treated with the indicated amounts of MG132. Sixteen hours posttreatment, cells were lysed and examined for ubiquitin, STAT2, STAT1, NS5, and GAPDH levels via Western blotting. (B) STAT2-deficient U6A cells were transfected with HA-ubiquitin, STAT2-FLAG, STAT1-GFP, NS2b-3, and either E- clv NS5-HA or an empty vector plasmid. Cells were then treated with lactacystin for 8 h and subsequently lysed and examined by Western blotting using ubiquitin, STAT2, STAT1, HA, and tubulin antibodies. (C) 293T cells were cotransfected with NS2b-3-HA and the plasmids indicated at the top. Ten hours posttransfection, cells were treated with the indicated amounts of lactacystin. Twenty-four hours posttransfection, cells were sorted for GFP-positive cells by FACS, lysed, and examined by Western blotting using ubiquitin-, GFP-, HA-, and GAPDH-specific antibodies. (D) STAT2-deficient U6A cells were transfected with 0.1 or 1 μg of FLAG-STAT2-FLAG, 0.1 μg NS2b-3, and either 1 μg E- clv NS5-HA or an empty vector plasmid. Cells were then lysed and examined by Western blotting using FLAG antibody and long-term film exposure to detect any additional low-intensity bands. The arrow indicates the expected size of FLAG-STAT2-FLAG. The asterisk marks a nonspecific band running at the same mobility of FLAG-STAT2-FLAG. (E) 293T cells were cotransfected with NS2b-3 and the plasmids indicated at the top. Twenty-four hours posttransfection, cells were sorted for GFP-positive cells by FACS, lysed, and examined via Western blotting using STAT2-, GFP-, HA-, FLAG-, and GAPDH-specific antibodies.

Techniques Used: Stable Transfection, Expressing, Western Blot, Transfection, Plasmid Preparation, FACS

DENV NS5 interacts with STAT2. (A) 293T cells were cotransfected with plasmids expressing FLAG-tagged STAT1 (STAT1-FLAG) and STAT2 (STAT2-FLAG) and empty plasmid (empty) or plasmid expressing HA-tagged DENV NS5 (NS5-HA), DENV core (CORE-HA), or NiV-V (HA-NiV-V) proteins. Lysates were then immunoprecipitated with anti-HA antibody (IP HA), and Western blotting was performed using anti-HA and anti-FLAG antibodies. Asterisks mark the heavy and light chains from the HA antibody. (B) 293T cells were cotransfected with plasmids expressing NS5-HA and either STAT1-FLAG or STAT2-FLAG. Lysates were then immunoprecipitated with anti-FLAG antibody (IP FLAG), and Western blotting was performed using anti-HA and anti-FLAG antibodies. TCE, total cell extracts were subjected to Western blotting using anti-HA, anti-FLAG, and anti-GAPDH antibodies.
Figure Legend Snippet: DENV NS5 interacts with STAT2. (A) 293T cells were cotransfected with plasmids expressing FLAG-tagged STAT1 (STAT1-FLAG) and STAT2 (STAT2-FLAG) and empty plasmid (empty) or plasmid expressing HA-tagged DENV NS5 (NS5-HA), DENV core (CORE-HA), or NiV-V (HA-NiV-V) proteins. Lysates were then immunoprecipitated with anti-HA antibody (IP HA), and Western blotting was performed using anti-HA and anti-FLAG antibodies. Asterisks mark the heavy and light chains from the HA antibody. (B) 293T cells were cotransfected with plasmids expressing NS5-HA and either STAT1-FLAG or STAT2-FLAG. Lysates were then immunoprecipitated with anti-FLAG antibody (IP FLAG), and Western blotting was performed using anti-HA and anti-FLAG antibodies. TCE, total cell extracts were subjected to Western blotting using anti-HA, anti-FLAG, and anti-GAPDH antibodies.

Techniques Used: Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot

Expression of a precursor form of DENV NS5 cleaved by the DENV protease results in reduced STAT2 levels. (A) 293T cells were transfected with the indicated constructs. Twenty-four hours posttransfection, cells were sorted for GFP-positive cells by FACS, subsequently lysed, and examined via Western blotting using GFP-, HA-, STAT1-, STAT2-, and GAPDH-specific antibodies. Schematics of the transfected constructs are shown at the bottom. ORFs that contain an “sp” at the N terminus have a signal peptide which directs the entire polyprotein to the surface of the ER for translation. Black arrows and red arrows indicate cleavage sites for cellular and the DEN2 viral proteases, respectively. The DEN2 active protease is highlighted in red. Densitometry analysis of the levels of STAT2 and NS5 are included on the far right, and levels are calculated relative to the levels in lane 1, with a value of 1 in the case of NS5 levels being indicative of no detection (background levels). (B) Same as above (A). Mutation of the DENV protease recognition site at the N terminus of NS5 is indicated by the blue dashes. The DENV protease labeled in blue indicates a serine-to-alanine mutation within the catalytic site of the DENV protease.
Figure Legend Snippet: Expression of a precursor form of DENV NS5 cleaved by the DENV protease results in reduced STAT2 levels. (A) 293T cells were transfected with the indicated constructs. Twenty-four hours posttransfection, cells were sorted for GFP-positive cells by FACS, subsequently lysed, and examined via Western blotting using GFP-, HA-, STAT1-, STAT2-, and GAPDH-specific antibodies. Schematics of the transfected constructs are shown at the bottom. ORFs that contain an “sp” at the N terminus have a signal peptide which directs the entire polyprotein to the surface of the ER for translation. Black arrows and red arrows indicate cleavage sites for cellular and the DEN2 viral proteases, respectively. The DEN2 active protease is highlighted in red. Densitometry analysis of the levels of STAT2 and NS5 are included on the far right, and levels are calculated relative to the levels in lane 1, with a value of 1 in the case of NS5 levels being indicative of no detection (background levels). (B) Same as above (A). Mutation of the DENV protease recognition site at the N terminus of NS5 is indicated by the blue dashes. The DENV protease labeled in blue indicates a serine-to-alanine mutation within the catalytic site of the DENV protease.

Techniques Used: Expressing, Transfection, Construct, FACS, Western Blot, Mutagenesis, Labeling

Cleaved NS5 is sufficient for reduced STAT2 levels and does not require an N-terminal glycine. (A) 293T cells were transfected with the indicated constructs. Twenty-four hours posttransfection, cells were sorted for GFP-positive cells by FACS, subsequently lysed, subjected to 4-to-20% SDS-polyacrylamide gel electrophoresis, and examined via Western blotting using GFP-, HA-, STAT1-, STAT2-, and GAPDH-specific antibodies. The TEV protease and its cleavage site are indicated by the green text and arrow, respectively. Cleavage sequences targeted by endogenous deubiquitinases are noted by a yellow arrow. Densitometry analyses of the levels of STAT2 and NS5 are included at the bottom, and levels were calculated relative to the levels in lane 1, with a value of 1 in the case of NS5 levels being indicative of no detection (background levels). (B) Same as above (A) except that lysates were run on a 7.5% SDS-polyacrylamide gel and subsequently analyzed by Western blotting using GFP-, STAT2-, and tubulin-specific antibodies.
Figure Legend Snippet: Cleaved NS5 is sufficient for reduced STAT2 levels and does not require an N-terminal glycine. (A) 293T cells were transfected with the indicated constructs. Twenty-four hours posttransfection, cells were sorted for GFP-positive cells by FACS, subsequently lysed, subjected to 4-to-20% SDS-polyacrylamide gel electrophoresis, and examined via Western blotting using GFP-, HA-, STAT1-, STAT2-, and GAPDH-specific antibodies. The TEV protease and its cleavage site are indicated by the green text and arrow, respectively. Cleavage sequences targeted by endogenous deubiquitinases are noted by a yellow arrow. Densitometry analyses of the levels of STAT2 and NS5 are included at the bottom, and levels were calculated relative to the levels in lane 1, with a value of 1 in the case of NS5 levels being indicative of no detection (background levels). (B) Same as above (A) except that lysates were run on a 7.5% SDS-polyacrylamide gel and subsequently analyzed by Western blotting using GFP-, STAT2-, and tubulin-specific antibodies.

Techniques Used: Transfection, Construct, FACS, Polyacrylamide Gel Electrophoresis, Western Blot

19) Product Images from "Myeloid derived suppressor cell inhibition of the IFN response in tumor-bearing mice"

Article Title: Myeloid derived suppressor cell inhibition of the IFN response in tumor-bearing mice

Journal: Cancer research

doi: 10.1158/0008-5472.CAN-10-2670

Elevated levels of nitric oxide (NO) in tumor bearing mice inhibits IFN responsiveness in immune cells (A) IF staining for iNOS protein (Fitc) and DAPI nuclear counterstain (left two panels). DAF-FM Fitc staining for nitric oxide and DAPI nuclear counterstain. (B) Splenocytes from normal mice were pre-treated with the nitrogen donor SNAP for 16 hours, stimulated for 15 minutes with PBS or IFN-A/D (10 4 U/mL) and evaluated for P-STAT1 by flow cytometry. (C) Protein lysates from splenocytes of normal or C26-bearing mice were immunoprecipitated with STAT1 antibody and then probed for nitrotyrosine. Lysates were also run on a separate gel and probed with an anti-STAT1 antibody (lower panel). Numbers represent densitometry values for nitrotyrosine and STAT1, and the ratio of nitrotyrosine/STAT1 (p=0.03). (D) P-STAT1 was measured in splenocytes obtained from day 21 iNOS knockout and control mice with and without C26 tumors by intracellular flow cytometry following stimulation with IFN-A/D (10 4 U/mL).
Figure Legend Snippet: Elevated levels of nitric oxide (NO) in tumor bearing mice inhibits IFN responsiveness in immune cells (A) IF staining for iNOS protein (Fitc) and DAPI nuclear counterstain (left two panels). DAF-FM Fitc staining for nitric oxide and DAPI nuclear counterstain. (B) Splenocytes from normal mice were pre-treated with the nitrogen donor SNAP for 16 hours, stimulated for 15 minutes with PBS or IFN-A/D (10 4 U/mL) and evaluated for P-STAT1 by flow cytometry. (C) Protein lysates from splenocytes of normal or C26-bearing mice were immunoprecipitated with STAT1 antibody and then probed for nitrotyrosine. Lysates were also run on a separate gel and probed with an anti-STAT1 antibody (lower panel). Numbers represent densitometry values for nitrotyrosine and STAT1, and the ratio of nitrotyrosine/STAT1 (p=0.03). (D) P-STAT1 was measured in splenocytes obtained from day 21 iNOS knockout and control mice with and without C26 tumors by intracellular flow cytometry following stimulation with IFN-A/D (10 4 U/mL).

Techniques Used: Mouse Assay, Staining, Flow Cytometry, Cytometry, Immunoprecipitation, Knock-Out

MDSC are elevated in tumor-bearing mice and are associated with decreased IFN responsiveness in vitro (A) Splenocytes from day 22 C26 mice were evaluated for MDSC using antibodies targeting GR1, IL-4Rα, and CD11b. (B) Splenocytes from day 22 C26 mice were evaluated for MDSC using antibodies targeting GR1 and CD11b. MDSC percentage in splenocytes from day 22 C26 tumor-bearing mice (C26; n = 19) or controls (Normal; n = 22) as described above. (C) MDSC were isolated from C26 splenocytes by magnetic labeling with anti-GR1 beads. After 24 hours, P-STAT1 was measured in splenocytes obtained from control mice (n = 10) or normal splenocytes that had been co-cultured with MDSC at a 1:3 ratio (n = 10).
Figure Legend Snippet: MDSC are elevated in tumor-bearing mice and are associated with decreased IFN responsiveness in vitro (A) Splenocytes from day 22 C26 mice were evaluated for MDSC using antibodies targeting GR1, IL-4Rα, and CD11b. (B) Splenocytes from day 22 C26 mice were evaluated for MDSC using antibodies targeting GR1 and CD11b. MDSC percentage in splenocytes from day 22 C26 tumor-bearing mice (C26; n = 19) or controls (Normal; n = 22) as described above. (C) MDSC were isolated from C26 splenocytes by magnetic labeling with anti-GR1 beads. After 24 hours, P-STAT1 was measured in splenocytes obtained from control mice (n = 10) or normal splenocytes that had been co-cultured with MDSC at a 1:3 ratio (n = 10).

Techniques Used: Mouse Assay, In Vitro, Isolation, Labeling, Cell Culture

Reduction in MDSC with gemcitabine or anti-GR1 restores IFN responsiveness (A) Splenocytes obtained from day 22 C26 tumor-bearing were evaluated by flow cytometry for the presence of MDSC. (B) P-STAT1 was measured in splenocytes obtained from control mice (Normal; n = 5), Day 22 mice bearing C26 tumors and treated with PBS (C26; n = 5), Day 22 mice bearing C26 tumors and treated with gemcitabine (C26+gemcitabine; n = 6), and Day 22 mice bearing C26 tumors and treated with gemcitabine 24-hours prior to harvest (C26+gemcitabine 24hr; n=5) by intracellular flow cytometry following stimulation with IFN-A/D (10 4 U/mL). (C) Splenocytes obtained from Day 21 control mice (Normal), mice bearing C26 tumors and treated with isotype control (C26+IgG; n = 6), or mice bearing C26 tumors and treated with anti-GR1 (C26+GR1; n = 7) were evaluated for MDSC by flow cytometry and (D) P-STAT1 was measured in splenocytes obtained from normal or day 21 tumor-bearing mice and treated with isotype control (C26+IgG; n = 6), or mice bearing C26 tumors and treated with anti-GR1 (C26+GR1; n = 7) by intracellular flow cytometry following a 15 minute stimulation with IFN-A/D (10 4 U/mL).
Figure Legend Snippet: Reduction in MDSC with gemcitabine or anti-GR1 restores IFN responsiveness (A) Splenocytes obtained from day 22 C26 tumor-bearing were evaluated by flow cytometry for the presence of MDSC. (B) P-STAT1 was measured in splenocytes obtained from control mice (Normal; n = 5), Day 22 mice bearing C26 tumors and treated with PBS (C26; n = 5), Day 22 mice bearing C26 tumors and treated with gemcitabine (C26+gemcitabine; n = 6), and Day 22 mice bearing C26 tumors and treated with gemcitabine 24-hours prior to harvest (C26+gemcitabine 24hr; n=5) by intracellular flow cytometry following stimulation with IFN-A/D (10 4 U/mL). (C) Splenocytes obtained from Day 21 control mice (Normal), mice bearing C26 tumors and treated with isotype control (C26+IgG; n = 6), or mice bearing C26 tumors and treated with anti-GR1 (C26+GR1; n = 7) were evaluated for MDSC by flow cytometry and (D) P-STAT1 was measured in splenocytes obtained from normal or day 21 tumor-bearing mice and treated with isotype control (C26+IgG; n = 6), or mice bearing C26 tumors and treated with anti-GR1 (C26+GR1; n = 7) by intracellular flow cytometry following a 15 minute stimulation with IFN-A/D (10 4 U/mL).

Techniques Used: Flow Cytometry, Cytometry, Mouse Assay

Decreased interferon response in tumor-bearing mice P-STAT1 was measured in splenocytes from control (Control; n = 19) or day 22 C26 tumor-bearing mice (C26 Tumor; n = 19) by intracellular flow cytometry following stimulation with (A) IFN-α (10 4 U/mL) or (B) IFN-γ (10 ng/mL). Y-axis is specific fluorescence (Fsp) of staining for P-STAT1. (C) P-STAT1 was measured in splenocytes from untreated normal mice and mice bearing C26 tumors, or from mice stimulated with 2×10 4 U IFN-A/D in vivo and harvested 2 hours later. (D) P-STAT1 was measured following IFN stimulation in splenocytes that were co-labeled with antibodies against CD4, CD8, and CD49b.
Figure Legend Snippet: Decreased interferon response in tumor-bearing mice P-STAT1 was measured in splenocytes from control (Control; n = 19) or day 22 C26 tumor-bearing mice (C26 Tumor; n = 19) by intracellular flow cytometry following stimulation with (A) IFN-α (10 4 U/mL) or (B) IFN-γ (10 ng/mL). Y-axis is specific fluorescence (Fsp) of staining for P-STAT1. (C) P-STAT1 was measured in splenocytes from untreated normal mice and mice bearing C26 tumors, or from mice stimulated with 2×10 4 U IFN-A/D in vivo and harvested 2 hours later. (D) P-STAT1 was measured following IFN stimulation in splenocytes that were co-labeled with antibodies against CD4, CD8, and CD49b.

Techniques Used: Mouse Assay, Flow Cytometry, Cytometry, Fluorescence, Staining, In Vivo, Labeling

20) Product Images from "Human metapneumovirus small hydrophobic (SH) protein downregulates type I IFN pathway signaling by affecting STAT1 expression and phosphorylation"

Article Title: Human metapneumovirus small hydrophobic (SH) protein downregulates type I IFN pathway signaling by affecting STAT1 expression and phosphorylation

Journal: Virology

doi: 10.1016/j.virol.2016.04.022

HMPV inhibits STAT1 phosphorylation of Vero cells only in infected cells. (A) Vero cells were infected for 24 h with HMPV at MOI of 1 in 6-well plates. Cells were lysed with RIPA buffer, run on 4-15% bis-tris gel, transferred to nitrocellulose membrane,
Figure Legend Snippet: HMPV inhibits STAT1 phosphorylation of Vero cells only in infected cells. (A) Vero cells were infected for 24 h with HMPV at MOI of 1 in 6-well plates. Cells were lysed with RIPA buffer, run on 4-15% bis-tris gel, transferred to nitrocellulose membrane,

Techniques Used: Infection

Type I IFN secreted by infected BEAS-2B cells inhibits STAT1 phosphorylation in neighboring uninfected cells. (A) BEAS-2B cells were infected with HMPV for 24 h at an MOI of 1 in 6-well plates, then stained with polyclonal anti-HMPV antibody and gated
Figure Legend Snippet: Type I IFN secreted by infected BEAS-2B cells inhibits STAT1 phosphorylation in neighboring uninfected cells. (A) BEAS-2B cells were infected with HMPV for 24 h at an MOI of 1 in 6-well plates, then stained with polyclonal anti-HMPV antibody and gated

Techniques Used: Infection, Staining

HMPV inhibits STAT1 phosphorylation in BEAS-2B cells. (A) BEAS-2B cells were infected for 24 h with HMPV at an MOI of 1 in 6-well plates. Cells were lysed with RIPA buffer, run on 4-15% bis-tris gel, transferred to nitrocellulose membrane, and probed
Figure Legend Snippet: HMPV inhibits STAT1 phosphorylation in BEAS-2B cells. (A) BEAS-2B cells were infected for 24 h with HMPV at an MOI of 1 in 6-well plates. Cells were lysed with RIPA buffer, run on 4-15% bis-tris gel, transferred to nitrocellulose membrane, and probed

Techniques Used: Infection

Transient expression of HMPV SH protein inhibits STAT1 phosphorylation. (A) Vero cells transfected with HMPV GFP-fusion proteins were analyzed compared to untransfected cells in the same culture using flow cytometry. (B) Transfected cells were analyzed
Figure Legend Snippet: Transient expression of HMPV SH protein inhibits STAT1 phosphorylation. (A) Vero cells transfected with HMPV GFP-fusion proteins were analyzed compared to untransfected cells in the same culture using flow cytometry. (B) Transfected cells were analyzed

Techniques Used: Expressing, Transfection, Flow Cytometry, Cytometry

21) Product Images from "The Orphan Nuclear Receptor TLX Is an Enhancer of STAT1-Mediated Transcription and Immunity to Toxoplasma gondiiTuning up STAT1"

Article Title: The Orphan Nuclear Receptor TLX Is an Enhancer of STAT1-Mediated Transcription and Immunity to Toxoplasma gondiiTuning up STAT1

Journal: PLoS Biology

doi: 10.1371/journal.pbio.1002200

TLX expression enhances occupancy of pSTAT1 on the CXCL9 and CXCL10 promoters. ChIP with rabbit monoclonal antibody to phosphorylated STAT1 (Y701) or control rabbit IgG. U2OS cells expressing either control vector or TLX were either left untreated or stimulated with IFN-γ for 2 h. The experiment was repeated twice with similar results. Data can be found in file S1 Data .
Figure Legend Snippet: TLX expression enhances occupancy of pSTAT1 on the CXCL9 and CXCL10 promoters. ChIP with rabbit monoclonal antibody to phosphorylated STAT1 (Y701) or control rabbit IgG. U2OS cells expressing either control vector or TLX were either left untreated or stimulated with IFN-γ for 2 h. The experiment was repeated twice with similar results. Data can be found in file S1 Data .

Techniques Used: Expressing, Chromatin Immunoprecipitation, Plasmid Preparation

TLX potentiation of STAT1 targets requires both the DNA binding domain and the ligand binding domain. (A) STAT1 reporter activity in unstimulated or IFN-γ-stimulated U2OS cells transduced with wild-type TLX (WT) or TLX truncation mutants consisting of the DNA binding domain only (∆DBD) or the ligand binding domain only (∆LBD). (B) RT-qPCR for CXCL9 and CXCL10 expression in U2OS cells transfected with WT TLX or truncation constructs. (C) RT-qPCRfor CXCL10 and OAS2 expression in unstimulated or IFN-γ-treated U251 astrocytes pretreated with famprofazone or DMSO. Mean and standard deviation is shown for three biological replicates. * = p ≤ 0.01. Experiment was repeated three times with similar results. Data for panels A, B, and C can be found in file S1 Data .
Figure Legend Snippet: TLX potentiation of STAT1 targets requires both the DNA binding domain and the ligand binding domain. (A) STAT1 reporter activity in unstimulated or IFN-γ-stimulated U2OS cells transduced with wild-type TLX (WT) or TLX truncation mutants consisting of the DNA binding domain only (∆DBD) or the ligand binding domain only (∆LBD). (B) RT-qPCR for CXCL9 and CXCL10 expression in U2OS cells transfected with WT TLX or truncation constructs. (C) RT-qPCRfor CXCL10 and OAS2 expression in unstimulated or IFN-γ-treated U251 astrocytes pretreated with famprofazone or DMSO. Mean and standard deviation is shown for three biological replicates. * = p ≤ 0.01. Experiment was repeated three times with similar results. Data for panels A, B, and C can be found in file S1 Data .

Techniques Used: Binding Assay, Ligand Binding Assay, Activity Assay, Transduction, Quantitative RT-PCR, Expressing, Transfection, Construct, Standard Deviation

STAT1 enhancers selectively potentiate the STAT1 pathway. Heatmap of showing row Z-scores based on log2 raw luminescent values for enhancers (right) tested on various signaling pathway reporters (top), either unstimulated (-) or stimulated with the cytokines shown on bottom (+). Values are averaged from three experiments. Color scheme legend is shown.
Figure Legend Snippet: STAT1 enhancers selectively potentiate the STAT1 pathway. Heatmap of showing row Z-scores based on log2 raw luminescent values for enhancers (right) tested on various signaling pathway reporters (top), either unstimulated (-) or stimulated with the cytokines shown on bottom (+). Values are averaged from three experiments. Color scheme legend is shown.

Techniques Used:

High-throughput screening identifies genes that restore function to the STAT1 pathway in Toxoplasma -infected cells. (A) Schematic showing workflow of STAT1 screen in Toxoplasma -infected cells. (B) Results of screen from experimental replicates, presented as fold change relative to infected cells transfected with an empty cDNA control vector. Uninfected cells transfected with empty cDNA controls are indicated in red, and infected cells transfected with MGC cDNA clones are in black. cDNAs that enhanced STAT1 activity ≥2.5-fold in both experiments are shown in the box in the upper right and inset (uninfected controls removed for clarity). (C) Protein domain family (Pfam)-based classification of nine validated cDNA hits that enhanced STAT1 activity ≥2.5-fold. (D) Protein–protein interaction network based on human orthologs of these hits (black) and STAT1 (red). Green indicates a network neighbor of STAT1. Data for panel B can be found in file S1 Data .
Figure Legend Snippet: High-throughput screening identifies genes that restore function to the STAT1 pathway in Toxoplasma -infected cells. (A) Schematic showing workflow of STAT1 screen in Toxoplasma -infected cells. (B) Results of screen from experimental replicates, presented as fold change relative to infected cells transfected with an empty cDNA control vector. Uninfected cells transfected with empty cDNA controls are indicated in red, and infected cells transfected with MGC cDNA clones are in black. cDNAs that enhanced STAT1 activity ≥2.5-fold in both experiments are shown in the box in the upper right and inset (uninfected controls removed for clarity). (C) Protein domain family (Pfam)-based classification of nine validated cDNA hits that enhanced STAT1 activity ≥2.5-fold. (D) Protein–protein interaction network based on human orthologs of these hits (black) and STAT1 (red). Green indicates a network neighbor of STAT1. Data for panel B can be found in file S1 Data .

Techniques Used: High Throughput Screening Assay, Infection, Transfection, Plasmid Preparation, Clone Assay, Activity Assay

22) Product Images from "IFN-λ4 potently blocks IFN-α signalling by ISG15 and USP18 in hepatitis C virus infection"

Article Title: IFN-λ4 potently blocks IFN-α signalling by ISG15 and USP18 in hepatitis C virus infection

Journal: Scientific Reports

doi: 10.1038/s41598-017-04186-7

IFN-λ4 overexpression induces ISG expression. ( A , B ) Huh-7 cells were transfected with control or IFN-λ4-expressing plasmids. After 72 hours, the cell lysates ( A ) and culture supernatants ( B ) were harvested, and protein expression were analysed by immunoblotting. For the culture supernatants, immunoprecipitation with anti-FLAG-conjugated beads were used to pull down the FLAG-tagged IFN-λ4 protein. The data are representative of two independent experiments. ( C , D ) Huh-7 cells were transfected with control, IFN-λ1-, IFN-λ4-, or IFN-β-expressing plasmids. After 96 hours, the cells were harvested, and protein expression or gene expression were analysed by immunoblotting ( C ) or real-time qPCR ( D ). ( E ) Huh-7 cells were transfected with IFN-λ4-expressing plasmids. The cells were harvested at the indicated time points, and gene expression was analysed by real-time qPCR. ( F ) Huh-7 cells were infected with JFH1 HCVcc at 0.5 MOI followed by transfection with control or IFN-λ4-expressing plasmids. After 72 hours, the cells were harvested, and HCV RNA titer was analysed by real-time qPCR. ( G , H ) Huh-7 cells were transfected with control, IFN-λ4-, or IFN-λ4 TT-encoding plasmids. After 96 hours, the cells were harvested, and protein and gene expression were analysed by immunoblotting ( G ) or real-time qPCR ( H ). In ( D , E , F , H ) data are presented as means ± S.E.M. *** P ≤ 0.001 (Student’s t-test). All the data are representative of two independent experiments. Full-length blots of PY-STAT1 ( C ) with multiple exposure times are included in the Supplementary Fig. 9C .
Figure Legend Snippet: IFN-λ4 overexpression induces ISG expression. ( A , B ) Huh-7 cells were transfected with control or IFN-λ4-expressing plasmids. After 72 hours, the cell lysates ( A ) and culture supernatants ( B ) were harvested, and protein expression were analysed by immunoblotting. For the culture supernatants, immunoprecipitation with anti-FLAG-conjugated beads were used to pull down the FLAG-tagged IFN-λ4 protein. The data are representative of two independent experiments. ( C , D ) Huh-7 cells were transfected with control, IFN-λ1-, IFN-λ4-, or IFN-β-expressing plasmids. After 96 hours, the cells were harvested, and protein expression or gene expression were analysed by immunoblotting ( C ) or real-time qPCR ( D ). ( E ) Huh-7 cells were transfected with IFN-λ4-expressing plasmids. The cells were harvested at the indicated time points, and gene expression was analysed by real-time qPCR. ( F ) Huh-7 cells were infected with JFH1 HCVcc at 0.5 MOI followed by transfection with control or IFN-λ4-expressing plasmids. After 72 hours, the cells were harvested, and HCV RNA titer was analysed by real-time qPCR. ( G , H ) Huh-7 cells were transfected with control, IFN-λ4-, or IFN-λ4 TT-encoding plasmids. After 96 hours, the cells were harvested, and protein and gene expression were analysed by immunoblotting ( G ) or real-time qPCR ( H ). In ( D , E , F , H ) data are presented as means ± S.E.M. *** P ≤ 0.001 (Student’s t-test). All the data are representative of two independent experiments. Full-length blots of PY-STAT1 ( C ) with multiple exposure times are included in the Supplementary Fig. 9C .

Techniques Used: Over Expression, Expressing, Transfection, Immunoprecipitation, Real-time Polymerase Chain Reaction, Infection

HCV infection results in IFN-λ4 expression and IFN-α unresponsiveness. ( A ) PHHs from 4 different donors were transfected with poly(I:C) (6 µg/ml). After 8 hours, the cells were harvested, and gene expression was analysed by real-time qPCR. ( B ) PHHs from 4 different donors were infected with JFH1 HCVcc at 10 MOI. After 48 hours, the cells were harvested, and gene expression was analysed by real-time qPCR. ( C ) PHHs with IFNL4 ΔG/TT genotype were infected with JFH1 HCVcc at 10 MOI. After 72 hours, the cell lysate was harvested, and protein expression were analysed by immunoblotting. ( D ) PHHs from two different donors (one with IFNL4 TT/TT genotype and the other with IFNL4 ΔG/ΔG genotype) were infected with JFH1 HCVcc at 10 MOI. After 72 hours, the cell lysates were harvested, and protein expression were analysed by immunoblotting. ( E ) PHHs with IFNL4 ΔG/TT genotype were infected with JFH1 HCVcc at 10 MOI. After 72 hours, the culture supernatant was harvested, and IFN-λ4 protein expression was analysed by immunoblotting. The data are representative of two independent experiments. ( F , G ) PHHs with the IFNL4 ΔG/ΔG genotype were infected with JFH1 HCVcc at 5 MOI. After 72 hours, the cells were treated with 100 IU/ml IFN-α for 30 minutes ( F ) or 6 hours ( G ). The cells were harvested, and gene and protein expression were analysed by immunoblotting ( F ) or real-time qPCR ( G ). Data are presented as means ± S.E.M. *** P ≤ 0.001 (Student’s t-test). All the data are representative of two independent experiments. Full-length blots of PY-STAT1 ( F ) with multiple exposure times are included in the Supplementary Fig. 9A .
Figure Legend Snippet: HCV infection results in IFN-λ4 expression and IFN-α unresponsiveness. ( A ) PHHs from 4 different donors were transfected with poly(I:C) (6 µg/ml). After 8 hours, the cells were harvested, and gene expression was analysed by real-time qPCR. ( B ) PHHs from 4 different donors were infected with JFH1 HCVcc at 10 MOI. After 48 hours, the cells were harvested, and gene expression was analysed by real-time qPCR. ( C ) PHHs with IFNL4 ΔG/TT genotype were infected with JFH1 HCVcc at 10 MOI. After 72 hours, the cell lysate was harvested, and protein expression were analysed by immunoblotting. ( D ) PHHs from two different donors (one with IFNL4 TT/TT genotype and the other with IFNL4 ΔG/ΔG genotype) were infected with JFH1 HCVcc at 10 MOI. After 72 hours, the cell lysates were harvested, and protein expression were analysed by immunoblotting. ( E ) PHHs with IFNL4 ΔG/TT genotype were infected with JFH1 HCVcc at 10 MOI. After 72 hours, the culture supernatant was harvested, and IFN-λ4 protein expression was analysed by immunoblotting. The data are representative of two independent experiments. ( F , G ) PHHs with the IFNL4 ΔG/ΔG genotype were infected with JFH1 HCVcc at 5 MOI. After 72 hours, the cells were treated with 100 IU/ml IFN-α for 30 minutes ( F ) or 6 hours ( G ). The cells were harvested, and gene and protein expression were analysed by immunoblotting ( F ) or real-time qPCR ( G ). Data are presented as means ± S.E.M. *** P ≤ 0.001 (Student’s t-test). All the data are representative of two independent experiments. Full-length blots of PY-STAT1 ( F ) with multiple exposure times are included in the Supplementary Fig. 9A .

Techniques Used: Infection, Expressing, Transfection, Real-time Polymerase Chain Reaction

HCV infection results in IFN-λ4 expression and IFN-α unresponsiveness that is restored by DAA treatment. ( A ) PHHs were infected with JFH1 HCVcc at 2 MOI. Shortly after HCV infection, telaprevir (5 μM) plus sofosbuvir (5 μM) were added to HCV-infected cells. After 24 or 48 hours, the cells were harvested, and gene expression was analysed by real-time qPCR. ( B ) PHHs with the IFNL4 ΔG/ΔG genotype were infected with JFH1 HCVcc at 2 MOI. Six hours after HCV infection, the culture medium was changed to the medium containing telaprevir (5 μM) and sofosbuvir (5 μM). After 120 hours, the cells were treated with 100 IU/ml of IFN-α for 30 minutes ( B ) or 6 hours ( C ). The cells were harvested, and gene and protein expression were analysed by immunoblotting ( B ) or real-time qPCR ( C ). Data are presented as means ± S.E.M. *** P ≤ 0.001 (Student’s t-test). DAA: direct-acting antivirals. All the data are representative of two independent experiments. Full-length blots of PY-STAT1 ( B ) with multiple exposure times are included in the Supplementary Fig. 9B . The data are representative of three independent experiments.
Figure Legend Snippet: HCV infection results in IFN-λ4 expression and IFN-α unresponsiveness that is restored by DAA treatment. ( A ) PHHs were infected with JFH1 HCVcc at 2 MOI. Shortly after HCV infection, telaprevir (5 μM) plus sofosbuvir (5 μM) were added to HCV-infected cells. After 24 or 48 hours, the cells were harvested, and gene expression was analysed by real-time qPCR. ( B ) PHHs with the IFNL4 ΔG/ΔG genotype were infected with JFH1 HCVcc at 2 MOI. Six hours after HCV infection, the culture medium was changed to the medium containing telaprevir (5 μM) and sofosbuvir (5 μM). After 120 hours, the cells were treated with 100 IU/ml of IFN-α for 30 minutes ( B ) or 6 hours ( C ). The cells were harvested, and gene and protein expression were analysed by immunoblotting ( B ) or real-time qPCR ( C ). Data are presented as means ± S.E.M. *** P ≤ 0.001 (Student’s t-test). DAA: direct-acting antivirals. All the data are representative of two independent experiments. Full-length blots of PY-STAT1 ( B ) with multiple exposure times are included in the Supplementary Fig. 9B . The data are representative of three independent experiments.

Techniques Used: Infection, Expressing, Real-time Polymerase Chain Reaction

23) Product Images from "Prolonged TNFα primes fibroblast-like synoviocytes in a gene-specific manner by altering chromatin"

Article Title: Prolonged TNFα primes fibroblast-like synoviocytes in a gene-specific manner by altering chromatin

Journal: Arthritis & rheumatology (Hoboken, N.J.)

doi: 10.1002/art.38871

STAT1 amplifier function of chronic TNFα in RA FLS RA FLS were cultured for 3 days in the presence or absence of TNFα (10 ng/ml), which was added on the first day of culture and was not replenished ( A, D ). ( A ), On day 3 the cells were stimulated with IFNβ (1,000 U/ml) or IFNγ (100 U/ml) for 10-60 minutes and STAT1 tyrosine phosphorylation (pY) was measured by immunoblotting. The expression of STAT1 protein ( B ) and mRNA ( C ), upon TNFα time course stimulation, was measured by immunoblotting and qPCR. Values are the mean ±SEM and were normalized relative to mRNA for GAPDH. *= p
Figure Legend Snippet: STAT1 amplifier function of chronic TNFα in RA FLS RA FLS were cultured for 3 days in the presence or absence of TNFα (10 ng/ml), which was added on the first day of culture and was not replenished ( A, D ). ( A ), On day 3 the cells were stimulated with IFNβ (1,000 U/ml) or IFNγ (100 U/ml) for 10-60 minutes and STAT1 tyrosine phosphorylation (pY) was measured by immunoblotting. The expression of STAT1 protein ( B ) and mRNA ( C ), upon TNFα time course stimulation, was measured by immunoblotting and qPCR. Values are the mean ±SEM and were normalized relative to mRNA for GAPDH. *= p

Techniques Used: Cell Culture, Expressing, Real-time Polymerase Chain Reaction

24) Product Images from "Structural analysis of the STAT1:STAT2 heterodimer revealed the mechanism of Sendai virus C protein–mediated blockade of type 1 interferon signaling"

Article Title: Structural analysis of the STAT1:STAT2 heterodimer revealed the mechanism of Sendai virus C protein–mediated blockade of type 1 interferon signaling

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M117.786285

Generation of N-terminal domain-deleted mutants of STAT1 and STAT2 and functional analysis of C proteins. A, schematic diagram of constructs of C′, C, Y1, Y2, and Y3. B, linear representation of the domains in human STAT1, STAT2, and their N-terminal domain-deleted mutants (STAT1ΔN and STAT2ΔN). N, N-terminal domain; CC, coiled-coil domain; DNA , DNA-binding domain; LK , linker domain; SH2 , SH2 domain; Y, phosphorylated tyrosine residue; TA , transcription activation domain. C, to estimate the strength of the response to IFN-α, subconfluent 293T cells were transfected with pISRE-EGFP and an expression plasmid for FL-C, FL-Y1, or FL-Y3, and IFN-α (20 units/ml) was added to the culture medium at 6 h after transfection. At 24 h post-transfection, photographs were taken under an immunofluorescent microscope. D, HeLa cells were transfected with an expression vector for HA-STAT1, HA-STAT2, or FL-C. A portion of the cell lysates prepared at 24 h post-transfection were mixed as indicated and immunoprecipitated ( IP ) with an anti-FLAG antibody ( Anti-FL ) together with protein G-Sepharose. The immunoprecipitates were separated by SDS-PAGE followed by Western blot analysis ( WB ) using an anti-HA antibody ( Anti-HA ) and anti-FL antibody. A part of the cell lysates was used to confirm protein expression using anti-HA and anti-FL antibodies. E, HeLa cells were transfected with an expression vector for HA-STAT1 or HA-STAT1ΔN together with an expression vector for FL-C or FL-Y3. At 24 h post-transfection, the cell lysate was immunoprecipitated with anti-FL antibody and analyzed by Western blotting using anti-HA and anti-FL antibodies. *, a light chain of IgG.
Figure Legend Snippet: Generation of N-terminal domain-deleted mutants of STAT1 and STAT2 and functional analysis of C proteins. A, schematic diagram of constructs of C′, C, Y1, Y2, and Y3. B, linear representation of the domains in human STAT1, STAT2, and their N-terminal domain-deleted mutants (STAT1ΔN and STAT2ΔN). N, N-terminal domain; CC, coiled-coil domain; DNA , DNA-binding domain; LK , linker domain; SH2 , SH2 domain; Y, phosphorylated tyrosine residue; TA , transcription activation domain. C, to estimate the strength of the response to IFN-α, subconfluent 293T cells were transfected with pISRE-EGFP and an expression plasmid for FL-C, FL-Y1, or FL-Y3, and IFN-α (20 units/ml) was added to the culture medium at 6 h after transfection. At 24 h post-transfection, photographs were taken under an immunofluorescent microscope. D, HeLa cells were transfected with an expression vector for HA-STAT1, HA-STAT2, or FL-C. A portion of the cell lysates prepared at 24 h post-transfection were mixed as indicated and immunoprecipitated ( IP ) with an anti-FLAG antibody ( Anti-FL ) together with protein G-Sepharose. The immunoprecipitates were separated by SDS-PAGE followed by Western blot analysis ( WB ) using an anti-HA antibody ( Anti-HA ) and anti-FL antibody. A part of the cell lysates was used to confirm protein expression using anti-HA and anti-FL antibodies. E, HeLa cells were transfected with an expression vector for HA-STAT1 or HA-STAT1ΔN together with an expression vector for FL-C or FL-Y3. At 24 h post-transfection, the cell lysate was immunoprecipitated with anti-FL antibody and analyzed by Western blotting using anti-HA and anti-FL antibodies. *, a light chain of IgG.

Techniques Used: Functional Assay, Construct, Binding Assay, Activation Assay, Transfection, Expressing, Plasmid Preparation, Microscopy, Immunoprecipitation, SDS Page, Western Blot

Inhibition of the phosphorylation of STAT2 with decreased affinity to STAT1ND in the presence of Y3. A, HeLa cells were transfected with an expression vector for FL-STAT2, FL-STAT2 R88E , or FL-STAT2 R88E-R92E together with an expression vector for wild-type HA-STAT2. At 24 h post-transfection, the cell lysate was immunoprecipitated with anti-FL antibody and analyzed by Western blotting using anti-HA and anti-FL antibodies. B, U6A cells were transfected with an expression vector for FL-STAT2, FL-STAT2 R88E , or FL-STAT2 R88E-R92E together with an expression vector for FL-Y3. At 24 h post-transfection, the cells were stimulated with IFN-α (1,000 units/ml) for 1 h ( B ). Proteins in the cell extracts were separated by SDS-PAGE for Western blot analysis using anti-pSTAT2, anti-FL, anti-STAT1, and anti-phosphorylated STAT1 antibodies. C, the rate of phosphorylation inhibition was determined on the basis of averaged signal intensity of FL-pSTAT2 in B , which was calculated from three independent experiments. Signal intensity of FL-STAT2 was used as an internal standard. D, relative quantities of phosphorylated STAT1 were determined on the basis of the average signal intensity of pSTAT1 in B , which was calculated from three independent experiments. Signal intensity of STAT1 was used as an internal standard.
Figure Legend Snippet: Inhibition of the phosphorylation of STAT2 with decreased affinity to STAT1ND in the presence of Y3. A, HeLa cells were transfected with an expression vector for FL-STAT2, FL-STAT2 R88E , or FL-STAT2 R88E-R92E together with an expression vector for wild-type HA-STAT2. At 24 h post-transfection, the cell lysate was immunoprecipitated with anti-FL antibody and analyzed by Western blotting using anti-HA and anti-FL antibodies. B, U6A cells were transfected with an expression vector for FL-STAT2, FL-STAT2 R88E , or FL-STAT2 R88E-R92E together with an expression vector for FL-Y3. At 24 h post-transfection, the cells were stimulated with IFN-α (1,000 units/ml) for 1 h ( B ). Proteins in the cell extracts were separated by SDS-PAGE for Western blot analysis using anti-pSTAT2, anti-FL, anti-STAT1, and anti-phosphorylated STAT1 antibodies. C, the rate of phosphorylation inhibition was determined on the basis of averaged signal intensity of FL-pSTAT2 in B , which was calculated from three independent experiments. Signal intensity of FL-STAT2 was used as an internal standard. D, relative quantities of phosphorylated STAT1 were determined on the basis of the average signal intensity of pSTAT1 in B , which was calculated from three independent experiments. Signal intensity of STAT1 was used as an internal standard.

Techniques Used: Inhibition, Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot, SDS Page

Inhibition of IFN-α-induced tyrosine phosphorylation of STAT1 in the presence of C protein. U3A cells were transfected with an expression vector for HA-STAT1 or HA-STAT1ΔN together with an expression vector for FL-C ( A ) or FL-Y3 ( B ). At 0.5 h after stimulation with IFN-α (1,000 units/ml), proteins in the cell extracts were separated by SDS-PAGE for Western blot analysis with an anti-STAT1 antibody ( anti-STAT1 ), an anti-Tyr 701 -phosphorylated STAT1 antibody ( anti-pSTAT1 ) and anti-FL. C, the rate of phosphorylation inhibition was determined on the basis of averaged signal intensities of HA-pSTAT1 in the presence or absence of FL-C ( A ) or FL-Y3 ( B ), which was calculated from three independent experiments. Intensities of bands were measured using ImageJ version 1.47, and signal intensity of HA-STAT1 was used as an internal standard. An error bar indicates standard deviation. p value was calculated on the basis of Welch's test.
Figure Legend Snippet: Inhibition of IFN-α-induced tyrosine phosphorylation of STAT1 in the presence of C protein. U3A cells were transfected with an expression vector for HA-STAT1 or HA-STAT1ΔN together with an expression vector for FL-C ( A ) or FL-Y3 ( B ). At 0.5 h after stimulation with IFN-α (1,000 units/ml), proteins in the cell extracts were separated by SDS-PAGE for Western blot analysis with an anti-STAT1 antibody ( anti-STAT1 ), an anti-Tyr 701 -phosphorylated STAT1 antibody ( anti-pSTAT1 ) and anti-FL. C, the rate of phosphorylation inhibition was determined on the basis of averaged signal intensities of HA-pSTAT1 in the presence or absence of FL-C ( A ) or FL-Y3 ( B ), which was calculated from three independent experiments. Intensities of bands were measured using ImageJ version 1.47, and signal intensity of HA-STAT1 was used as an internal standard. An error bar indicates standard deviation. p value was calculated on the basis of Welch's test.

Techniques Used: Inhibition, Transfection, Expressing, Plasmid Preparation, SDS Page, Western Blot, Standard Deviation

25) Product Images from "The NF-κB p65 and p50 homodimer cooperate with IRF8 to activate iNOS transcription"

Article Title: The NF-κB p65 and p50 homodimer cooperate with IRF8 to activate iNOS transcription

Journal: BMC Cancer

doi: 10.1186/s12885-015-1808-6

IFNγ and NF-κB induce iNOS expression in myeloid cells. a J774 cells were treated with IFNγ, LPS, or both IFNγ and LPS for approximately 18 h, and analyzed for iNOS expression by RT-PCR. β-actin was used as a normalization control. b Cells were treated as in A and then analyzed by real time RT-PCR analysis of iNOS expression with β-action as an internal control. c J774 cells were cultured in the presence of Ruxolitinib for 30 min and then treated with IFNγ and LPS as indicated for 18 h. Total lysates were then prepared and analyzed for STAT1 and pSTAT1 levels by Western blotting analysis. d J774 cells were cultured in the presence of Ruxolitinib for 30 min and then treated with IFNγ and LPS for 18 h. iNOS expression was then analyzed by RT-PCR. e J774 cells were transiently transfected with a control vector or a vector containing the dominant negative IκBα-AA mutant, respectively. Cells were treated with IFNγ and LPS for approximately 18 h, and then analyzed for iNOS expression
Figure Legend Snippet: IFNγ and NF-κB induce iNOS expression in myeloid cells. a J774 cells were treated with IFNγ, LPS, or both IFNγ and LPS for approximately 18 h, and analyzed for iNOS expression by RT-PCR. β-actin was used as a normalization control. b Cells were treated as in A and then analyzed by real time RT-PCR analysis of iNOS expression with β-action as an internal control. c J774 cells were cultured in the presence of Ruxolitinib for 30 min and then treated with IFNγ and LPS as indicated for 18 h. Total lysates were then prepared and analyzed for STAT1 and pSTAT1 levels by Western blotting analysis. d J774 cells were cultured in the presence of Ruxolitinib for 30 min and then treated with IFNγ and LPS for 18 h. iNOS expression was then analyzed by RT-PCR. e J774 cells were transiently transfected with a control vector or a vector containing the dominant negative IκBα-AA mutant, respectively. Cells were treated with IFNγ and LPS for approximately 18 h, and then analyzed for iNOS expression

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Cell Culture, Western Blot, Transfection, Plasmid Preparation, Dominant Negative Mutation, Mutagenesis

IFNγ and TNFα cooperatively induce iNOS expression in human colon carcinoma cells. a Tumor cells were treated with IFNγ, TNFα, or both IFNγ and TNFα for approximately 18 h, and analyzed for iNOS expression by RT-PCR. β-actin was used as a normalization control. b Cells were treated as in A and then analyzed by Western blotting analysis of iNOS expression with β-action as an internal control. c Tumor cells were cultured in the presence of Ruxolitinib for 30 min and then treated with IFNγ and TNFα as indicated for 18 h. Total lysates were then prepared and analyzed for STAT1 and pSTAT1 levels by Western blotting analysis. d The cells were treated as in C and then analyzed by RT-PCR for iNOS expression. e The cells were transfected with either scramble siRNA or human IRF8-specific siRNA for 6 h and the cells were treated with IFNγ for 18 h. The cells were analyzed for IRF8 and iNOS expression by RT-PCR with β-actin as a normalization control
Figure Legend Snippet: IFNγ and TNFα cooperatively induce iNOS expression in human colon carcinoma cells. a Tumor cells were treated with IFNγ, TNFα, or both IFNγ and TNFα for approximately 18 h, and analyzed for iNOS expression by RT-PCR. β-actin was used as a normalization control. b Cells were treated as in A and then analyzed by Western blotting analysis of iNOS expression with β-action as an internal control. c Tumor cells were cultured in the presence of Ruxolitinib for 30 min and then treated with IFNγ and TNFα as indicated for 18 h. Total lysates were then prepared and analyzed for STAT1 and pSTAT1 levels by Western blotting analysis. d The cells were treated as in C and then analyzed by RT-PCR for iNOS expression. e The cells were transfected with either scramble siRNA or human IRF8-specific siRNA for 6 h and the cells were treated with IFNγ for 18 h. The cells were analyzed for IRF8 and iNOS expression by RT-PCR with β-actin as a normalization control

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Cell Culture, Transfection

26) Product Images from "A novel form of human STAT1 deficiency impairing early but not late responses to interferons"

Article Title: A novel form of human STAT1 deficiency impairing early but not late responses to interferons

Journal: Blood

doi: 10.1182/blood-2010-04-280586

Abnormal mRNA splicing resulting from K201N . (A) RT-PCR of a full-length STAT1 α, STAT1 β, STAT1 fragment running from exon 7 to exon 10, and glyceraldehyde-3-phosphate dehydrogenase from mRNA extracted from the EBV-B cells of a healthy
Figure Legend Snippet: Abnormal mRNA splicing resulting from K201N . (A) RT-PCR of a full-length STAT1 α, STAT1 β, STAT1 fragment running from exon 7 to exon 10, and glyceraldehyde-3-phosphate dehydrogenase from mRNA extracted from the EBV-B cells of a healthy

Techniques Used: Reverse Transcription Polymerase Chain Reaction

STAT1 protein expression is impaired in the cells of P1 . EBV-B cells from WT/WT, P1, K201N/WT, 1928insA/WT (WT/−), P696S/P696S, and 1928insA/1928insA (−/−) persons were stimulated with 10 5 IU/mL IFN-α or IFN-γ or
Figure Legend Snippet: STAT1 protein expression is impaired in the cells of P1 . EBV-B cells from WT/WT, P1, K201N/WT, 1928insA/WT (WT/−), P696S/P696S, and 1928insA/1928insA (−/−) persons were stimulated with 10 5 IU/mL IFN-α or IFN-γ or

Techniques Used: Expressing

K201N mutation does not impair STAT1 dephosphorylation and homodimerization . (A) K201N does not impair homodimerization. U3C cells were transfected with a combination of mock (M), WT, and K201N-mutated STAT1 plasmids tagged with either Flag or Myc. Proteins
Figure Legend Snippet: K201N mutation does not impair STAT1 dephosphorylation and homodimerization . (A) K201N does not impair homodimerization. U3C cells were transfected with a combination of mock (M), WT, and K201N-mutated STAT1 plasmids tagged with either Flag or Myc. Proteins

Techniques Used: Mutagenesis, De-Phosphorylation Assay, Transfection

The STAT1 K201N mutation caused susceptibility to mycobacterial and viral infections . (A) The human STAT1 coding region with its known mutations. Coding exons are numbered with Roman numerals and delineated by vertical bars. The N-terminal domain, coiled-coil
Figure Legend Snippet: The STAT1 K201N mutation caused susceptibility to mycobacterial and viral infections . (A) The human STAT1 coding region with its known mutations. Coding exons are numbered with Roman numerals and delineated by vertical bars. The N-terminal domain, coiled-coil

Techniques Used: Mutagenesis

27) Product Images from "Interferon-free treatment for hepatitis C virus infection induces normalization of extrahepatic type I interferon signaling"

Article Title: Interferon-free treatment for hepatitis C virus infection induces normalization of extrahepatic type I interferon signaling

Journal: Clinical and Molecular Hepatology

doi: 10.3350/cmh.2017.0074

STAT1 phosphorylation decreased in the peripheral blood mononuclear cells (PBMCs) of HCV-infected patients after direct-acting antiviral (DAA) treatment. (A, C) PBMCs from HCV-infected patients were isolated before DAA (pre DAA) and at the end of DAA (post DAA) treatment. Immunoblotting was performed to detect the protein levels of STAT1, PY-STAT1, and GAPDH. (B) PBMCs from HCV-infected patients were isolated before DAA treatment. TaqMan real-time quantitative polymerase chain reaction was performed to detect mRNA levels of IFI44, CXCL10, and GAPDH. Pearson's correlation analysis was performed to identify the associations between expression levels of IFI44/CXCL10 and relative band intensity of PY-STAT1. STAT1, signal transducer and activator of transcription 1; HCV, hepatitis C virus; PY-STAT1, tyrosine-phosphorylated STAT1; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IFI44, interferon-induced protein 44; CXCL10, C-X-C motif chemokine ligand 10. * P
Figure Legend Snippet: STAT1 phosphorylation decreased in the peripheral blood mononuclear cells (PBMCs) of HCV-infected patients after direct-acting antiviral (DAA) treatment. (A, C) PBMCs from HCV-infected patients were isolated before DAA (pre DAA) and at the end of DAA (post DAA) treatment. Immunoblotting was performed to detect the protein levels of STAT1, PY-STAT1, and GAPDH. (B) PBMCs from HCV-infected patients were isolated before DAA treatment. TaqMan real-time quantitative polymerase chain reaction was performed to detect mRNA levels of IFI44, CXCL10, and GAPDH. Pearson's correlation analysis was performed to identify the associations between expression levels of IFI44/CXCL10 and relative band intensity of PY-STAT1. STAT1, signal transducer and activator of transcription 1; HCV, hepatitis C virus; PY-STAT1, tyrosine-phosphorylated STAT1; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IFI44, interferon-induced protein 44; CXCL10, C-X-C motif chemokine ligand 10. * P

Techniques Used: Infection, Isolation, Real-time Polymerase Chain Reaction, Expressing

28) Product Images from "Rapid Inflammation in Mice Lacking Both SOCS1 and SOCS3 in Hematopoietic Cells"

Article Title: Rapid Inflammation in Mice Lacking Both SOCS1 and SOCS3 in Hematopoietic Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0162111

Interleukin-6 signalling in mice lacking SOCS proteins. Western blot analysis of macrophages prepared from mice 14d after tamoxifen or vehicle treatment. The cells were stimulated with IL-6 and lysates were prepared at the times indicated. Proteins were separated by polyacrylamide gel electrophoresis, transferred to membranes and probed with antibodies to the molecules indicated at the right. Replicate filters were prepared from the same lysates and probed with pSTAT1 and pSTAT3 and subsequently with STAT1 and STAT3 and SOCS3. Images were scanned and cropped to prepare the Figure; for uncropped images see S6 Fig .
Figure Legend Snippet: Interleukin-6 signalling in mice lacking SOCS proteins. Western blot analysis of macrophages prepared from mice 14d after tamoxifen or vehicle treatment. The cells were stimulated with IL-6 and lysates were prepared at the times indicated. Proteins were separated by polyacrylamide gel electrophoresis, transferred to membranes and probed with antibodies to the molecules indicated at the right. Replicate filters were prepared from the same lysates and probed with pSTAT1 and pSTAT3 and subsequently with STAT1 and STAT3 and SOCS3. Images were scanned and cropped to prepare the Figure; for uncropped images see S6 Fig .

Techniques Used: Mouse Assay, Western Blot, Polyacrylamide Gel Electrophoresis

29) Product Images from "Human metapneumovirus small hydrophobic (SH) protein downregulates type I IFN pathway signaling by affecting STAT1 expression and phosphorylation"

Article Title: Human metapneumovirus small hydrophobic (SH) protein downregulates type I IFN pathway signaling by affecting STAT1 expression and phosphorylation

Journal: Virology

doi: 10.1016/j.virol.2016.04.022

HMPV inhibits STAT1 phosphorylation of Vero cells only in infected cells. (A) Vero cells were infected for 24 h with HMPV at MOI of 1 in 6-well plates. Cells were lysed with RIPA buffer, run on 4-15% bis-tris gel, transferred to nitrocellulose membrane,
Figure Legend Snippet: HMPV inhibits STAT1 phosphorylation of Vero cells only in infected cells. (A) Vero cells were infected for 24 h with HMPV at MOI of 1 in 6-well plates. Cells were lysed with RIPA buffer, run on 4-15% bis-tris gel, transferred to nitrocellulose membrane,

Techniques Used: Infection

Type I IFN secreted by infected BEAS-2B cells inhibits STAT1 phosphorylation in neighboring uninfected cells. (A) BEAS-2B cells were infected with HMPV for 24 h at an MOI of 1 in 6-well plates, then stained with polyclonal anti-HMPV antibody and gated
Figure Legend Snippet: Type I IFN secreted by infected BEAS-2B cells inhibits STAT1 phosphorylation in neighboring uninfected cells. (A) BEAS-2B cells were infected with HMPV for 24 h at an MOI of 1 in 6-well plates, then stained with polyclonal anti-HMPV antibody and gated

Techniques Used: Infection, Staining

HMPV inhibits STAT1 phosphorylation in BEAS-2B cells. (A) BEAS-2B cells were infected for 24 h with HMPV at an MOI of 1 in 6-well plates. Cells were lysed with RIPA buffer, run on 4-15% bis-tris gel, transferred to nitrocellulose membrane, and probed
Figure Legend Snippet: HMPV inhibits STAT1 phosphorylation in BEAS-2B cells. (A) BEAS-2B cells were infected for 24 h with HMPV at an MOI of 1 in 6-well plates. Cells were lysed with RIPA buffer, run on 4-15% bis-tris gel, transferred to nitrocellulose membrane, and probed

Techniques Used: Infection

Transient expression of HMPV SH protein inhibits STAT1 phosphorylation. (A) Vero cells transfected with HMPV GFP-fusion proteins were analyzed compared to untransfected cells in the same culture using flow cytometry. (B) Transfected cells were analyzed
Figure Legend Snippet: Transient expression of HMPV SH protein inhibits STAT1 phosphorylation. (A) Vero cells transfected with HMPV GFP-fusion proteins were analyzed compared to untransfected cells in the same culture using flow cytometry. (B) Transfected cells were analyzed

Techniques Used: Expressing, Transfection, Flow Cytometry, Cytometry

30) Product Images from "Human metapneumovirus small hydrophobic (SH) protein downregulates type I IFN pathway signaling by affecting STAT1 expression and phosphorylation"

Article Title: Human metapneumovirus small hydrophobic (SH) protein downregulates type I IFN pathway signaling by affecting STAT1 expression and phosphorylation

Journal: Virology

doi: 10.1016/j.virol.2016.04.022

HMPV inhibits STAT1 phosphorylation of Vero cells only in infected cells. (A) Vero cells were infected for 24 h with HMPV at MOI of 1 in 6-well plates. Cells were lysed with RIPA buffer, run on 4-15% bis-tris gel, transferred to nitrocellulose membrane,
Figure Legend Snippet: HMPV inhibits STAT1 phosphorylation of Vero cells only in infected cells. (A) Vero cells were infected for 24 h with HMPV at MOI of 1 in 6-well plates. Cells were lysed with RIPA buffer, run on 4-15% bis-tris gel, transferred to nitrocellulose membrane,

Techniques Used: Infection

Type I IFN secreted by infected BEAS-2B cells inhibits STAT1 phosphorylation in neighboring uninfected cells. (A) BEAS-2B cells were infected with HMPV for 24 h at an MOI of 1 in 6-well plates, then stained with polyclonal anti-HMPV antibody and gated
Figure Legend Snippet: Type I IFN secreted by infected BEAS-2B cells inhibits STAT1 phosphorylation in neighboring uninfected cells. (A) BEAS-2B cells were infected with HMPV for 24 h at an MOI of 1 in 6-well plates, then stained with polyclonal anti-HMPV antibody and gated

Techniques Used: Infection, Staining

HMPV inhibits STAT1 phosphorylation in BEAS-2B cells. (A) BEAS-2B cells were infected for 24 h with HMPV at an MOI of 1 in 6-well plates. Cells were lysed with RIPA buffer, run on 4-15% bis-tris gel, transferred to nitrocellulose membrane, and probed
Figure Legend Snippet: HMPV inhibits STAT1 phosphorylation in BEAS-2B cells. (A) BEAS-2B cells were infected for 24 h with HMPV at an MOI of 1 in 6-well plates. Cells were lysed with RIPA buffer, run on 4-15% bis-tris gel, transferred to nitrocellulose membrane, and probed

Techniques Used: Infection

Transient expression of HMPV SH protein inhibits STAT1 phosphorylation. (A) Vero cells transfected with HMPV GFP-fusion proteins were analyzed compared to untransfected cells in the same culture using flow cytometry. (B) Transfected cells were analyzed
Figure Legend Snippet: Transient expression of HMPV SH protein inhibits STAT1 phosphorylation. (A) Vero cells transfected with HMPV GFP-fusion proteins were analyzed compared to untransfected cells in the same culture using flow cytometry. (B) Transfected cells were analyzed

Techniques Used: Expressing, Transfection, Flow Cytometry, Cytometry

31) Product Images from "Human metapneumovirus small hydrophobic (SH) protein downregulates type I IFN pathway signaling by affecting STAT1 expression and phosphorylation"

Article Title: Human metapneumovirus small hydrophobic (SH) protein downregulates type I IFN pathway signaling by affecting STAT1 expression and phosphorylation

Journal: Virology

doi: 10.1016/j.virol.2016.04.022

HMPV inhibits STAT1 phosphorylation of Vero cells only in infected cells. (A) Vero cells were infected for 24 h with HMPV at MOI of 1 in 6-well plates. Cells were lysed with RIPA buffer, run on 4-15% bis-tris gel, transferred to nitrocellulose membrane,
Figure Legend Snippet: HMPV inhibits STAT1 phosphorylation of Vero cells only in infected cells. (A) Vero cells were infected for 24 h with HMPV at MOI of 1 in 6-well plates. Cells were lysed with RIPA buffer, run on 4-15% bis-tris gel, transferred to nitrocellulose membrane,

Techniques Used: Infection

Type I IFN secreted by infected BEAS-2B cells inhibits STAT1 phosphorylation in neighboring uninfected cells. (A) BEAS-2B cells were infected with HMPV for 24 h at an MOI of 1 in 6-well plates, then stained with polyclonal anti-HMPV antibody and gated
Figure Legend Snippet: Type I IFN secreted by infected BEAS-2B cells inhibits STAT1 phosphorylation in neighboring uninfected cells. (A) BEAS-2B cells were infected with HMPV for 24 h at an MOI of 1 in 6-well plates, then stained with polyclonal anti-HMPV antibody and gated

Techniques Used: Infection, Staining

HMPV inhibits STAT1 phosphorylation in BEAS-2B cells. (A) BEAS-2B cells were infected for 24 h with HMPV at an MOI of 1 in 6-well plates. Cells were lysed with RIPA buffer, run on 4-15% bis-tris gel, transferred to nitrocellulose membrane, and probed
Figure Legend Snippet: HMPV inhibits STAT1 phosphorylation in BEAS-2B cells. (A) BEAS-2B cells were infected for 24 h with HMPV at an MOI of 1 in 6-well plates. Cells were lysed with RIPA buffer, run on 4-15% bis-tris gel, transferred to nitrocellulose membrane, and probed

Techniques Used: Infection

Transient expression of HMPV SH protein inhibits STAT1 phosphorylation. (A) Vero cells transfected with HMPV GFP-fusion proteins were analyzed compared to untransfected cells in the same culture using flow cytometry. (B) Transfected cells were analyzed
Figure Legend Snippet: Transient expression of HMPV SH protein inhibits STAT1 phosphorylation. (A) Vero cells transfected with HMPV GFP-fusion proteins were analyzed compared to untransfected cells in the same culture using flow cytometry. (B) Transfected cells were analyzed

Techniques Used: Expressing, Transfection, Flow Cytometry, Cytometry

32) Product Images from "Human metapneumovirus small hydrophobic (SH) protein downregulates type I IFN pathway signaling by affecting STAT1 expression and phosphorylation"

Article Title: Human metapneumovirus small hydrophobic (SH) protein downregulates type I IFN pathway signaling by affecting STAT1 expression and phosphorylation

Journal: Virology

doi: 10.1016/j.virol.2016.04.022

HMPV inhibits STAT1 phosphorylation of Vero cells only in infected cells. (A) Vero cells were infected for 24 h with HMPV at MOI of 1 in 6-well plates. Cells were lysed with RIPA buffer, run on 4-15% bis-tris gel, transferred to nitrocellulose membrane,
Figure Legend Snippet: HMPV inhibits STAT1 phosphorylation of Vero cells only in infected cells. (A) Vero cells were infected for 24 h with HMPV at MOI of 1 in 6-well plates. Cells were lysed with RIPA buffer, run on 4-15% bis-tris gel, transferred to nitrocellulose membrane,

Techniques Used: Infection

Type I IFN secreted by infected BEAS-2B cells inhibits STAT1 phosphorylation in neighboring uninfected cells. (A) BEAS-2B cells were infected with HMPV for 24 h at an MOI of 1 in 6-well plates, then stained with polyclonal anti-HMPV antibody and gated
Figure Legend Snippet: Type I IFN secreted by infected BEAS-2B cells inhibits STAT1 phosphorylation in neighboring uninfected cells. (A) BEAS-2B cells were infected with HMPV for 24 h at an MOI of 1 in 6-well plates, then stained with polyclonal anti-HMPV antibody and gated

Techniques Used: Infection, Staining

HMPV inhibits STAT1 phosphorylation in BEAS-2B cells. (A) BEAS-2B cells were infected for 24 h with HMPV at an MOI of 1 in 6-well plates. Cells were lysed with RIPA buffer, run on 4-15% bis-tris gel, transferred to nitrocellulose membrane, and probed
Figure Legend Snippet: HMPV inhibits STAT1 phosphorylation in BEAS-2B cells. (A) BEAS-2B cells were infected for 24 h with HMPV at an MOI of 1 in 6-well plates. Cells were lysed with RIPA buffer, run on 4-15% bis-tris gel, transferred to nitrocellulose membrane, and probed

Techniques Used: Infection

Transient expression of HMPV SH protein inhibits STAT1 phosphorylation. (A) Vero cells transfected with HMPV GFP-fusion proteins were analyzed compared to untransfected cells in the same culture using flow cytometry. (B) Transfected cells were analyzed
Figure Legend Snippet: Transient expression of HMPV SH protein inhibits STAT1 phosphorylation. (A) Vero cells transfected with HMPV GFP-fusion proteins were analyzed compared to untransfected cells in the same culture using flow cytometry. (B) Transfected cells were analyzed

Techniques Used: Expressing, Transfection, Flow Cytometry, Cytometry

33) Product Images from "EBP1 protein modulates the expression of human MHC class II molecules in non-hematopoietic cancer cells"

Article Title: EBP1 protein modulates the expression of human MHC class II molecules in non-hematopoietic cancer cells

Journal: International Journal of Oncology

doi: 10.3892/ijo.2015.3051

Analysis of STAT1 phosphorylation and mRNA decay in U87, MCF7 and M14 transfectants. Western blot analysis of MCF7, U87 and M14 cell extracts overexpressing p42 and p48 isoforms performed by antip-STAT1. Anti-β-tubulin was used to normalize the amount of proteins.
Figure Legend Snippet: Analysis of STAT1 phosphorylation and mRNA decay in U87, MCF7 and M14 transfectants. Western blot analysis of MCF7, U87 and M14 cell extracts overexpressing p42 and p48 isoforms performed by antip-STAT1. Anti-β-tubulin was used to normalize the amount of proteins.

Techniques Used: Western Blot

34) Product Images from "Indirect Inhibition of Toll-like Receptor and Type I Interferon Responses by ITAM-coupled Receptors and Integrins"

Article Title: Indirect Inhibition of Toll-like Receptor and Type I Interferon Responses by ITAM-coupled Receptors and Integrins

Journal: Immunity

doi: 10.1016/j.immuni.2010.03.014

β2 Integrin Ligation by Fb Inhibits IFN-α and TLR Signaling (A) Immunoblot analysis of whole cell lysates of primary human macrophages that were kept suspended (Non-adh) or added to plates coated with fibrinogen (Fb) for the indicated times, and then stimulated with IFN-α (25 ng/ml) for 15 minutes. Blots were probed with antibodies against tyrosine-phosphorylated STAT1 (pY-STAT1), tyrosine-phosphorylated STAT3 (pY-STAT3), STAT1 and STAT3. Data are representative of 15 similar experiments. (B) Real time RT-PCR analysis of CXCL9 mRNA from primary human macrophages plated on Fb-coated plates for the indicated times and stimulated with IFN-α (25 ng/ml) for 3 hr. Results are presented as mean +/- s.d. of triplicate wells normalized relative to GAPDH mRNA and are representative of at least 3 experiments. (C) Real time RT-PCR analysis of IL-6 and TNF mRNA from primary human macrophages plated on Fb-coated plates for 1 hr, followed by stimulation with LPS (10 ng/ml) or Pam3Cys (10 ng/ml) for 3 hours. Results are presented as mean +/- s.d. of triplicate wells normalized relative to GAPDH mRNA and are representative of at least 3 experiments. .
Figure Legend Snippet: β2 Integrin Ligation by Fb Inhibits IFN-α and TLR Signaling (A) Immunoblot analysis of whole cell lysates of primary human macrophages that were kept suspended (Non-adh) or added to plates coated with fibrinogen (Fb) for the indicated times, and then stimulated with IFN-α (25 ng/ml) for 15 minutes. Blots were probed with antibodies against tyrosine-phosphorylated STAT1 (pY-STAT1), tyrosine-phosphorylated STAT3 (pY-STAT3), STAT1 and STAT3. Data are representative of 15 similar experiments. (B) Real time RT-PCR analysis of CXCL9 mRNA from primary human macrophages plated on Fb-coated plates for the indicated times and stimulated with IFN-α (25 ng/ml) for 3 hr. Results are presented as mean +/- s.d. of triplicate wells normalized relative to GAPDH mRNA and are representative of at least 3 experiments. (C) Real time RT-PCR analysis of IL-6 and TNF mRNA from primary human macrophages plated on Fb-coated plates for 1 hr, followed by stimulation with LPS (10 ng/ml) or Pam3Cys (10 ng/ml) for 3 hours. Results are presented as mean +/- s.d. of triplicate wells normalized relative to GAPDH mRNA and are representative of at least 3 experiments. .

Techniques Used: Ligation, Quantitative RT-PCR

35) Product Images from "MicroRNA-155 controls CD8+ T cell responses by regulating interferon signaling"

Article Title: MicroRNA-155 controls CD8+ T cell responses by regulating interferon signaling

Journal: Nature immunology

doi: 10.1038/ni.2576

miR-155 regulates STAT1 expression and Type I IFN signaling contributes to the proliferative defect of miR-155 deficiency. (a) and (b) Increased total STAT1 in miR-155-KO OT-I cells. (a) Representative histogram showing STAT1 expression and (b) fold increase of STAT1 MFI in vitro in miR-155-KO OT-I cells over OT-I cells. Cells activated with OVA(257–264)-pulsed irradiated splenocytes for 4 days (data from 5 experiments). * P
Figure Legend Snippet: miR-155 regulates STAT1 expression and Type I IFN signaling contributes to the proliferative defect of miR-155 deficiency. (a) and (b) Increased total STAT1 in miR-155-KO OT-I cells. (a) Representative histogram showing STAT1 expression and (b) fold increase of STAT1 MFI in vitro in miR-155-KO OT-I cells over OT-I cells. Cells activated with OVA(257–264)-pulsed irradiated splenocytes for 4 days (data from 5 experiments). * P

Techniques Used: Expressing, In Vitro, Irradiation

36) Product Images from "Inhibition of p38 Mitogen-activated Protein Kinase Impairs Influenza Virus-induced Primary and Secondary Host Gene Responses and Protects Mice from Lethal H5N1 Infection *"

Article Title: Inhibition of p38 Mitogen-activated Protein Kinase Impairs Influenza Virus-induced Primary and Secondary Host Gene Responses and Protects Mice from Lethal H5N1 Infection *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M113.469239

p38 MAPK inhibition affects H5N1-induced IFN expression. A, impact of p38 MAPK inhibition on the IFNβ promoter activity. Vero cells were transfected with the IFNβ promoter for 24 h. Cells were preincubated with 20 μ m SB 202190 or left untreated and subsequently stimulated with 500 ng of total RNA isolated from infected A549 cells (8 h, 5 m.o.i.). Total RNA from uninfected A549 cells was used as control. 5 h p.s. promoter activity was measured by a luciferase assay and the results are depicted as mean n -fold (± S.D.) of three independent experiments normalized to controls. B, Western blot analysis of total lysates of HUVEC treated with UV-inactivated, filtered conditioned media from mock-infected control cells ( lanes 5 and 7 ) and KAN-1-infected cells (5 m.o.i., 5 h) ( lanes 6 and 8 ). Donor cells were pretreated with DMSO ( lanes 1 and 3 ) or SB 202190 (20 μ m , lanes 2 and 4 ). STAT1 Tyr 701 phosphorylation was detected 15 min after treatment with conditioned medium ( upper panel ). Equal loading was verified by the detection of total ERK2 ( lower panel ). Blots are representative of three independent experiments.
Figure Legend Snippet: p38 MAPK inhibition affects H5N1-induced IFN expression. A, impact of p38 MAPK inhibition on the IFNβ promoter activity. Vero cells were transfected with the IFNβ promoter for 24 h. Cells were preincubated with 20 μ m SB 202190 or left untreated and subsequently stimulated with 500 ng of total RNA isolated from infected A549 cells (8 h, 5 m.o.i.). Total RNA from uninfected A549 cells was used as control. 5 h p.s. promoter activity was measured by a luciferase assay and the results are depicted as mean n -fold (± S.D.) of three independent experiments normalized to controls. B, Western blot analysis of total lysates of HUVEC treated with UV-inactivated, filtered conditioned media from mock-infected control cells ( lanes 5 and 7 ) and KAN-1-infected cells (5 m.o.i., 5 h) ( lanes 6 and 8 ). Donor cells were pretreated with DMSO ( lanes 1 and 3 ) or SB 202190 (20 μ m , lanes 2 and 4 ). STAT1 Tyr 701 phosphorylation was detected 15 min after treatment with conditioned medium ( upper panel ). Equal loading was verified by the detection of total ERK2 ( lower panel ). Blots are representative of three independent experiments.

Techniques Used: Inhibition, Expressing, Activity Assay, Transfection, Isolation, Infection, Luciferase, Western Blot

Influenza A virus infection induces phosphorylation of STAT1 on serine 727. A, Western blot analysis of total lysates of A549 cells infected with 5 m.o.i. of FPV or KAN-1. P-STAT1 Ser 727 ( upper panel ) and p-p38 ( middle panel ) were detected 4–8 h post-infection. Equal loading was verified by the detection of total STAT1 and total p38. B and D, left: A549 ( B ) or Vero cells ( D ) were preincubated with 5 or 10 μ m SB 202190 in comparison to DMSO. Cells were infected with 5 m.o.i. of FPV and incubated with the respective concentrations of SB 202190 for the indicated time points. Right, A549 ( B ) or Vero cells ( D ) were preincubated with 10 μ m SB 202190 or SB 203580 in comparison to DMSO. Cells were subsequently infected with FPV (5 m.o.i.) for the indicated time points. C and E, left, A549 ( C ) or Vero cells ( E ) were transfected with p38 α-specific siRNA. 48 h post-transfection cells were infected with FPV (5 m.o.i.) for 8 h. Efficient siRNA-mediated knockdown was confirmed by p38α detection. B–E , P-STAT1 Ser 727 ( upper panel ) and viral proteins PB2 and M1 ( middle panels ) were detected by Western blot analysis. Equal loading was verified by the detection of total STAT1 and total ERK2. C and E, right, densitometric analysis of FPV-induced STAT1 Ser 727 phosphorylation levels in the presence or absence of p38 α siRNA. Phosphorylation levels were estimated as the relative intensity of the appropriate phosphorylation bands to the loading control normalized to the appropriate uninfected controls. Intensities are depicted as mean ± S.D. of three independent experiments. F, Vero cells were preincubated with 20 μ m SB 202190 or DMSO and subsequently infected with FPV (1 m.o.i.) for 9 h. Viral titers are depicted as mean ± S.D. of three independent experiments. G, expression of STAT1 Y701F and double mutant (STAT1 Y701F/S727) in A549 cells was confirmed by Western blot analysis ( upper panel , lanes 2 and 3 ). Equal loading was confirmed by the detection of total ERK2. A-E and G, blots are representative of three independent experiments. H and I , A549 cells stably expressing STAT1 Y701F, STAT1 Y701F/S727A, or the empty vector were transfected with the different ISG promoters, as indicated. Cells were stimulated with 500 units/ml of recombinant human IFNβ ( H , ISRE) or IFNγ ( I , GAS). 8 h post-stimulation promoter activity was measured and the results are depicted as mean n -fold (± S.D.) of three independent experiments normalized to STAT1 Y701F activity.
Figure Legend Snippet: Influenza A virus infection induces phosphorylation of STAT1 on serine 727. A, Western blot analysis of total lysates of A549 cells infected with 5 m.o.i. of FPV or KAN-1. P-STAT1 Ser 727 ( upper panel ) and p-p38 ( middle panel ) were detected 4–8 h post-infection. Equal loading was verified by the detection of total STAT1 and total p38. B and D, left: A549 ( B ) or Vero cells ( D ) were preincubated with 5 or 10 μ m SB 202190 in comparison to DMSO. Cells were infected with 5 m.o.i. of FPV and incubated with the respective concentrations of SB 202190 for the indicated time points. Right, A549 ( B ) or Vero cells ( D ) were preincubated with 10 μ m SB 202190 or SB 203580 in comparison to DMSO. Cells were subsequently infected with FPV (5 m.o.i.) for the indicated time points. C and E, left, A549 ( C ) or Vero cells ( E ) were transfected with p38 α-specific siRNA. 48 h post-transfection cells were infected with FPV (5 m.o.i.) for 8 h. Efficient siRNA-mediated knockdown was confirmed by p38α detection. B–E , P-STAT1 Ser 727 ( upper panel ) and viral proteins PB2 and M1 ( middle panels ) were detected by Western blot analysis. Equal loading was verified by the detection of total STAT1 and total ERK2. C and E, right, densitometric analysis of FPV-induced STAT1 Ser 727 phosphorylation levels in the presence or absence of p38 α siRNA. Phosphorylation levels were estimated as the relative intensity of the appropriate phosphorylation bands to the loading control normalized to the appropriate uninfected controls. Intensities are depicted as mean ± S.D. of three independent experiments. F, Vero cells were preincubated with 20 μ m SB 202190 or DMSO and subsequently infected with FPV (1 m.o.i.) for 9 h. Viral titers are depicted as mean ± S.D. of three independent experiments. G, expression of STAT1 Y701F and double mutant (STAT1 Y701F/S727) in A549 cells was confirmed by Western blot analysis ( upper panel , lanes 2 and 3 ). Equal loading was confirmed by the detection of total ERK2. A-E and G, blots are representative of three independent experiments. H and I , A549 cells stably expressing STAT1 Y701F, STAT1 Y701F/S727A, or the empty vector were transfected with the different ISG promoters, as indicated. Cells were stimulated with 500 units/ml of recombinant human IFNβ ( H , ISRE) or IFNγ ( I , GAS). 8 h post-stimulation promoter activity was measured and the results are depicted as mean n -fold (± S.D.) of three independent experiments normalized to STAT1 Y701F activity.

Techniques Used: Infection, Western Blot, Incubation, Transfection, Expressing, Mutagenesis, Stable Transfection, Plasmid Preparation, Recombinant, Activity Assay

37) Product Images from "Unphosphorylated STAT1 promotes sarcoma development through repressing expression of Fas and Bad and conferring apoptotic resistance"

Article Title: Unphosphorylated STAT1 promotes sarcoma development through repressing expression of Fas and Bad and conferring apoptotic resistance

Journal: Cancer research

doi: 10.1158/0008-5472.CAN-12-1347

STAT1 regulates Bcl-xL, Fas and Bad expression A-C . CMS4 cells were transfected with scramble and mouse STAT1-specific siRNAs, respectively, overnight, and analyzed for the expression level of genes as indicated by semi-quantitative RT-PCR. Cell surface Fas protein levels were measured by staining cells with Fas-specific mAb and analyzed by flow cytometry. Fas protein level is quantified by mean fluorescence intensity and presented in bottom of panel C. * p =0.05. D . Bcl-xL and Bad form a protein complex in sarcoma cells. Cell lysates were prepared from CMS4 cells, immunoprecipitated with Bcl-2- and Bcl-xL-specific mAbs, respectively, and then analyzed for Bcl-2, Bcl-xL and Bad protein association by Western blotting analysis.
Figure Legend Snippet: STAT1 regulates Bcl-xL, Fas and Bad expression A-C . CMS4 cells were transfected with scramble and mouse STAT1-specific siRNAs, respectively, overnight, and analyzed for the expression level of genes as indicated by semi-quantitative RT-PCR. Cell surface Fas protein levels were measured by staining cells with Fas-specific mAb and analyzed by flow cytometry. Fas protein level is quantified by mean fluorescence intensity and presented in bottom of panel C. * p =0.05. D . Bcl-xL and Bad form a protein complex in sarcoma cells. Cell lysates were prepared from CMS4 cells, immunoprecipitated with Bcl-2- and Bcl-xL-specific mAbs, respectively, and then analyzed for Bcl-2, Bcl-xL and Bad protein association by Western blotting analysis.

Techniques Used: Expressing, Transfection, Quantitative RT-PCR, Staining, Flow Cytometry, Cytometry, Fluorescence, Immunoprecipitation, Western Blot

uSTAT1 represses Jak1 expression to inhibit IFN-γ-induced STAT1 phosphorylation A . Disruption of IRF8 function results in diminished pSTAT1 and Jak1 level in sarcoma cells. The indicated 4 cell sublines were cultured in the presence and absence of IFN-γ for 24 h and analyzed for pSTAT1 and Jak1 protein levels by Western blotting analysis. B . Disruption of IRF8 function results in diminished pSTAT1 activity in sarcoma cells. CMS4 cells were cultured in the presence or absence of IFN-γ for 4 h. Nuclear extracts were prepared from the cells and incubated with a GAS element-containing DNA probe. The protein-DNA interactions were then analyzed by EMSA. C . STAT1 is a repressor of Jak1. CMS4 cells were transfected with scramble and mouse STAT1-specific siRNAs, respectively, overnight and analyzed for STAT1 and Jak1 expression level by semi-quantitative RT-PCR. D . IFN-γ-induced STAT1 activation (pSTAT1) and expression (uSTAT1) kinetics in sarcoma cells. CMS4 cells were cultured in the presence of IFN-γ for the indicated time and analyzed for pSTAT1 and total STAT1 protein level by Western blotting analysis.
Figure Legend Snippet: uSTAT1 represses Jak1 expression to inhibit IFN-γ-induced STAT1 phosphorylation A . Disruption of IRF8 function results in diminished pSTAT1 and Jak1 level in sarcoma cells. The indicated 4 cell sublines were cultured in the presence and absence of IFN-γ for 24 h and analyzed for pSTAT1 and Jak1 protein levels by Western blotting analysis. B . Disruption of IRF8 function results in diminished pSTAT1 activity in sarcoma cells. CMS4 cells were cultured in the presence or absence of IFN-γ for 4 h. Nuclear extracts were prepared from the cells and incubated with a GAS element-containing DNA probe. The protein-DNA interactions were then analyzed by EMSA. C . STAT1 is a repressor of Jak1. CMS4 cells were transfected with scramble and mouse STAT1-specific siRNAs, respectively, overnight and analyzed for STAT1 and Jak1 expression level by semi-quantitative RT-PCR. D . IFN-γ-induced STAT1 activation (pSTAT1) and expression (uSTAT1) kinetics in sarcoma cells. CMS4 cells were cultured in the presence of IFN-γ for the indicated time and analyzed for pSTAT1 and total STAT1 protein level by Western blotting analysis.

Techniques Used: Expressing, Cell Culture, Western Blot, Activity Assay, Incubation, Transfection, Quantitative RT-PCR, Activation Assay

STAT1 immunohistochemistry on human sarcoma Human STS TMA slides were stained with STAT1 mAb as described in the Materials and Methods. Shown are examples of low (A, C, E, G I: 40X) and high (B, D J: 200X; F H: 400x) power STAT1 staining of strong (A,B) and weak (C,D) cytoplasmic staining, strong (E,F) and weak (G,H) nuclear staining, and negative (I,J) staiing in tumor cells. Infiltrating T-cell lymphocytes serve as a positive internal control (highlighted in panel I J) and there identity was confirmed using CD3 immunohistochemistry.
Figure Legend Snippet: STAT1 immunohistochemistry on human sarcoma Human STS TMA slides were stained with STAT1 mAb as described in the Materials and Methods. Shown are examples of low (A, C, E, G I: 40X) and high (B, D J: 200X; F H: 400x) power STAT1 staining of strong (A,B) and weak (C,D) cytoplasmic staining, strong (E,F) and weak (G,H) nuclear staining, and negative (I,J) staiing in tumor cells. Infiltrating T-cell lymphocytes serve as a positive internal control (highlighted in panel I J) and there identity was confirmed using CD3 immunohistochemistry.

Techniques Used: Immunohistochemistry, Staining

Correlation analysis between STAT1 protein level in tumor cells and disease-specific survival A. Kaplan-Meier survival curve for disease-specific survival (DSS) between nSTAT1-positive and negative tumors. STS specimens from 123 STS patients were stained with STAT1-specific mAb and examined for nSTAT1 protein in the tumor cells. The nSTAT1-positive and negative groups were then analyzed for correlation with DSS. B . Kaplan-Meier survival curve for DSS and correlation with cSTAT1 protein level in the tumor cells. STS specimens were analyzed as in A and cSTAT1 high protein level group was compared to the cSTAT1 low protein level group for correlation with DSS. C . The IRF8 promoter DNA is hypermethylated in human STS. Genomic DNA was isolated from dissected human STS cells and treated with bisulfite. The modified DNA was then analyzed with MS-PCR primers specific for the human IRF8 promoter. U: unmethylation; M: methylation.
Figure Legend Snippet: Correlation analysis between STAT1 protein level in tumor cells and disease-specific survival A. Kaplan-Meier survival curve for disease-specific survival (DSS) between nSTAT1-positive and negative tumors. STS specimens from 123 STS patients were stained with STAT1-specific mAb and examined for nSTAT1 protein in the tumor cells. The nSTAT1-positive and negative groups were then analyzed for correlation with DSS. B . Kaplan-Meier survival curve for DSS and correlation with cSTAT1 protein level in the tumor cells. STS specimens were analyzed as in A and cSTAT1 high protein level group was compared to the cSTAT1 low protein level group for correlation with DSS. C . The IRF8 promoter DNA is hypermethylated in human STS. Genomic DNA was isolated from dissected human STS cells and treated with bisulfite. The modified DNA was then analyzed with MS-PCR primers specific for the human IRF8 promoter. U: unmethylation; M: methylation.

Techniques Used: Staining, Isolation, Modification, Mass Spectrometry, Polymerase Chain Reaction, Methylation

Silencing STAT1 expression increases sarcoma cell sensitivity to Fas-mediated apoptosis in vitro A . CMS4 cells were transfected with scramble and mouse STAT1-specific siRNAs, respectively, overnight, and analyzed for STAT1 expression level by semi-quantitative RT-PCR (left panel) and real-time RT-PCR (right panel). B . The STAT1 siRNA-transfected cells were incubated in the absence or presence of FasL (250 ng/ml) overnight and then stained with PI and Annexin V. The stained cells were analyzed with flow cytometry. The number in each plot indicated the PI and Annexin V double positive cells. Apoptotic cell death was quantified by the formula: percentage of PI + and Annexin V + cells in the presence of FasL - percentage of PI + and Annexin V + cells without FasL treatment, and presented at the right panel.
Figure Legend Snippet: Silencing STAT1 expression increases sarcoma cell sensitivity to Fas-mediated apoptosis in vitro A . CMS4 cells were transfected with scramble and mouse STAT1-specific siRNAs, respectively, overnight, and analyzed for STAT1 expression level by semi-quantitative RT-PCR (left panel) and real-time RT-PCR (right panel). B . The STAT1 siRNA-transfected cells were incubated in the absence or presence of FasL (250 ng/ml) overnight and then stained with PI and Annexin V. The stained cells were analyzed with flow cytometry. The number in each plot indicated the PI and Annexin V double positive cells. Apoptotic cell death was quantified by the formula: percentage of PI + and Annexin V + cells in the presence of FasL - percentage of PI + and Annexin V + cells without FasL treatment, and presented at the right panel.

Techniques Used: Expressing, In Vitro, Transfection, Quantitative RT-PCR, Incubation, Staining, Flow Cytometry, Cytometry

38) Product Images from "Promoter Occupancy of STAT1 in Interferon Responses Is Regulated by Processive Transcription"

Article Title: Promoter Occupancy of STAT1 in Interferon Responses Is Regulated by Processive Transcription

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.01097-14

ActD and flavopiridol inhibit STAT1 dephosphorylation independently of p53- and miRNA-mediated mechanisms. (A) Transcription inhibition by flavopiridol (FP) impairs STAT1 and STAT2 tyrosine dephosphorylation. BMDMs were either left untreated or pretreated
Figure Legend Snippet: ActD and flavopiridol inhibit STAT1 dephosphorylation independently of p53- and miRNA-mediated mechanisms. (A) Transcription inhibition by flavopiridol (FP) impairs STAT1 and STAT2 tyrosine dephosphorylation. BMDMs were either left untreated or pretreated

Techniques Used: De-Phosphorylation Assay, Inhibition

Inhibition of transcription is permissive for Y701 dephosphorylation of nucleoplasmic STAT1. (A) STAT1 and STAT2 tyrosine dephosphorylation in the absence of Irf9. WT and Irf9 −/− BMDMs were stimulated with IFN-β for the indicated
Figure Legend Snippet: Inhibition of transcription is permissive for Y701 dephosphorylation of nucleoplasmic STAT1. (A) STAT1 and STAT2 tyrosine dephosphorylation in the absence of Irf9. WT and Irf9 −/− BMDMs were stimulated with IFN-β for the indicated

Techniques Used: Inhibition, De-Phosphorylation Assay

Analysis of IFN-β-induced gene expression and STAT1 promoter occupancy in BMDMs expressing solely the STAT1β isoform. (A and B) IFN-β-induced transcription in WT and STAT1β BMDMs. Cells were stimulated with IFN-β;
Figure Legend Snippet: Analysis of IFN-β-induced gene expression and STAT1 promoter occupancy in BMDMs expressing solely the STAT1β isoform. (A and B) IFN-β-induced transcription in WT and STAT1β BMDMs. Cells were stimulated with IFN-β;

Techniques Used: Expressing

Transcription inhibition by ActD prolongs STAT1 but not NF-κB occupancy at target promoters. (A and B) Transcription inhibition prolongs STAT1 occupancy at the Irf1 and Mx2 promoters. BMDMs were stimulated with IFN-β in the presence or
Figure Legend Snippet: Transcription inhibition by ActD prolongs STAT1 but not NF-κB occupancy at target promoters. (A and B) Transcription inhibition prolongs STAT1 occupancy at the Irf1 and Mx2 promoters. BMDMs were stimulated with IFN-β in the presence or

Techniques Used: Inhibition

Tyrosine dephosphorylation of IFN-activated STAT1 is dependent on ongoing transcription. (A) Kinetics of STAT1 Y701 dephosphorylation after type I IFN stimulation. BMDMs were stimulated with IFN-β for the indicated times, and cell extracts were
Figure Legend Snippet: Tyrosine dephosphorylation of IFN-activated STAT1 is dependent on ongoing transcription. (A) Kinetics of STAT1 Y701 dephosphorylation after type I IFN stimulation. BMDMs were stimulated with IFN-β for the indicated times, and cell extracts were

Techniques Used: De-Phosphorylation Assay

Processive transcription is required for downregulation of STAT1 promoter occupancy but not for Y701 dephosphorylation. (A) Pervanadate (Na 3 VO 4 ) inhibits the tyrosine dephosphorylation of STAT1 and STAT2. BMDMs were stimulated with IFN-β in the
Figure Legend Snippet: Processive transcription is required for downregulation of STAT1 promoter occupancy but not for Y701 dephosphorylation. (A) Pervanadate (Na 3 VO 4 ) inhibits the tyrosine dephosphorylation of STAT1 and STAT2. BMDMs were stimulated with IFN-β in the

Techniques Used: De-Phosphorylation Assay

Transcription inhibition by flavopiridol and DRB prolongs STAT1 promoter occupancy. (A and B) Flavopiridol (FP) inhibits IFN-β-induced transcription. BMDMs that had been left untreated (w/o) or pretreated with FP for 15 min were stimulated with
Figure Legend Snippet: Transcription inhibition by flavopiridol and DRB prolongs STAT1 promoter occupancy. (A and B) Flavopiridol (FP) inhibits IFN-β-induced transcription. BMDMs that had been left untreated (w/o) or pretreated with FP for 15 min were stimulated with

Techniques Used: Inhibition

39) Product Images from "The small molecule curcumin analog FLLL32 induces apoptosis in melanoma cells via STAT3 inhibition and retains the cellular response to cytokines with anti-tumor activity"

Article Title: The small molecule curcumin analog FLLL32 induces apoptosis in melanoma cells via STAT3 inhibition and retains the cellular response to cytokines with anti-tumor activity

Journal: Molecular Cancer

doi: 10.1186/1476-4598-9-165

IFN-γ-induced signal transduction was not adversely affected by FLLL32 . (A) A375 cells were pre-treated for 16 hours with FLLL32 (2 -- 14 μM) or curcumin (20 μM) and subsequently treated with IFN-γ (10 ng/mL) for 15 minutes. IFN-γ-induced pSTAT1 and pSTAT3 were evaluated by immunoblot. Total STAT1, STAT3 and β-actin were also measured to control for loading. The data were also summarized by densitometry comparing relative expression of pSTAT1 to STAT1 and pSTAT3 to STAT3. (B) The same experiment was performed whereby A375 cells were pre-treated for 16 hours with other Jak2/STAT3 pathway inhibitors (WP1066, JSI-124, Stattic) prior to IFN-γ stimulation. Data shown are representative of two separate experiments and similar results were obtained in the Hs294T human melanoma cell line (Additional File 1 : Figure S3). (C) IFN-γ-induced gene expression was enhanced in the presence of FLLL32. A375, Hs294T or 1106 MEL cells were pre-treated for 1 hour with 2μM FLLL32, 20μM curcumin or DMSO (negative control), and subsequently stimulated with IFN-γ (10 ng/mL) or PBS (vehicle) for an additional 4 hours. Expression of IRF1 was evaluated by Real Time PCR. Data were normalized to 18s rRNA levels (housekeeping gene) and expressed as the mean fold change versus DMSO-pre-treated cells stimulated with PBS. Error bars represent the standard deviation from n = 2 independent experiments.
Figure Legend Snippet: IFN-γ-induced signal transduction was not adversely affected by FLLL32 . (A) A375 cells were pre-treated for 16 hours with FLLL32 (2 -- 14 μM) or curcumin (20 μM) and subsequently treated with IFN-γ (10 ng/mL) for 15 minutes. IFN-γ-induced pSTAT1 and pSTAT3 were evaluated by immunoblot. Total STAT1, STAT3 and β-actin were also measured to control for loading. The data were also summarized by densitometry comparing relative expression of pSTAT1 to STAT1 and pSTAT3 to STAT3. (B) The same experiment was performed whereby A375 cells were pre-treated for 16 hours with other Jak2/STAT3 pathway inhibitors (WP1066, JSI-124, Stattic) prior to IFN-γ stimulation. Data shown are representative of two separate experiments and similar results were obtained in the Hs294T human melanoma cell line (Additional File 1 : Figure S3). (C) IFN-γ-induced gene expression was enhanced in the presence of FLLL32. A375, Hs294T or 1106 MEL cells were pre-treated for 1 hour with 2μM FLLL32, 20μM curcumin or DMSO (negative control), and subsequently stimulated with IFN-γ (10 ng/mL) or PBS (vehicle) for an additional 4 hours. Expression of IRF1 was evaluated by Real Time PCR. Data were normalized to 18s rRNA levels (housekeeping gene) and expressed as the mean fold change versus DMSO-pre-treated cells stimulated with PBS. Error bars represent the standard deviation from n = 2 independent experiments.

Techniques Used: Transduction, Expressing, Negative Control, Real-time Polymerase Chain Reaction, Standard Deviation

40) Product Images from "NS5 of Dengue Virus Mediates STAT2 Binding and Degradation ▿"

Article Title: NS5 of Dengue Virus Mediates STAT2 Binding and Degradation ▿

Journal: Journal of Virology

doi: 10.1128/JVI.02188-08

Truncations in the DENV NS5 protein affect its ability to associate with STAT2, decrease STAT2 levels, and inhibit IFN signaling. (A) 293T cells were transfected with the indicated constructs. Numbering refers to the glycine start position within the NS5 protein. All numbered forms of NS5 are expressed within the context of the E-Ub-NS5-GFP construct (i.e., 10-900, NS5 residues 10 to 900 fused to the C terminus of the E-Ub cassette). Twenty-four hours posttransfection, cells were sorted for GFP-positive cells using FACS, lysed, and examined by Western blotting using STAT2, GFP, and tubulin antibodies. (B) 293T cells were cotransfected with the indicated plasmids (the numbering system is identical to that described in A), STAT1-FLAG, and STAT2-FLAG. In order to detect STAT2 binding by NS5, STAT2-FLAG plasmid was transfected in excess with respect to E-Ub-NS5-GFP plasmids, resulting in a not-detectable degradation of STAT2-FLAG. Lysates were immunoprecipitated with a polyclonal GFP antibody (pGFP), and Western blots were performed using FLAG, GFP, and GAPDH antibodies. TCE, total cell extracts. (C) 293T cells were transfected with the ISRE-54-CAT reporter, a constitutively expressing firefly luciferase plasmid, and the indicated HA-tagged viral protein. Twenty-four hours posttransfection, cells were treated with 1,000 U/ml of type I IFN. Twenty-four hours posttreatment, cells were lysed and measured for CAT and luciferase activity. Data are represented with the standard deviations from three independent experiments. Samples showing P values of less than 0.05 compared with the empty control sample are indicated.
Figure Legend Snippet: Truncations in the DENV NS5 protein affect its ability to associate with STAT2, decrease STAT2 levels, and inhibit IFN signaling. (A) 293T cells were transfected with the indicated constructs. Numbering refers to the glycine start position within the NS5 protein. All numbered forms of NS5 are expressed within the context of the E-Ub-NS5-GFP construct (i.e., 10-900, NS5 residues 10 to 900 fused to the C terminus of the E-Ub cassette). Twenty-four hours posttransfection, cells were sorted for GFP-positive cells using FACS, lysed, and examined by Western blotting using STAT2, GFP, and tubulin antibodies. (B) 293T cells were cotransfected with the indicated plasmids (the numbering system is identical to that described in A), STAT1-FLAG, and STAT2-FLAG. In order to detect STAT2 binding by NS5, STAT2-FLAG plasmid was transfected in excess with respect to E-Ub-NS5-GFP plasmids, resulting in a not-detectable degradation of STAT2-FLAG. Lysates were immunoprecipitated with a polyclonal GFP antibody (pGFP), and Western blots were performed using FLAG, GFP, and GAPDH antibodies. TCE, total cell extracts. (C) 293T cells were transfected with the ISRE-54-CAT reporter, a constitutively expressing firefly luciferase plasmid, and the indicated HA-tagged viral protein. Twenty-four hours posttransfection, cells were treated with 1,000 U/ml of type I IFN. Twenty-four hours posttreatment, cells were lysed and measured for CAT and luciferase activity. Data are represented with the standard deviations from three independent experiments. Samples showing P values of less than 0.05 compared with the empty control sample are indicated.

Techniques Used: Transfection, Construct, FACS, Western Blot, Binding Assay, Plasmid Preparation, Immunoprecipitation, Expressing, Luciferase, Activity Assay

Inhibitors of the ubiquitin-proteasome pathway prevent STAT2 degradation by DENV NS5. (A) wtVero or Vero cells stably expressing the DEN1 replicon were treated with the indicated amounts of MG132. Sixteen hours posttreatment, cells were lysed and examined for ubiquitin, STAT2, STAT1, NS5, and GAPDH levels via Western blotting. (B) STAT2-deficient U6A cells were transfected with HA-ubiquitin, STAT2-FLAG, STAT1-GFP, NS2b-3, and either E- clv NS5-HA or an empty vector plasmid. Cells were then treated with lactacystin for 8 h and subsequently lysed and examined by Western blotting using ubiquitin, STAT2, STAT1, HA, and tubulin antibodies. (C) 293T cells were cotransfected with NS2b-3-HA and the plasmids indicated at the top. Ten hours posttransfection, cells were treated with the indicated amounts of lactacystin. Twenty-four hours posttransfection, cells were sorted for GFP-positive cells by FACS, lysed, and examined by Western blotting using ubiquitin-, GFP-, HA-, and GAPDH-specific antibodies. (D) STAT2-deficient U6A cells were transfected with 0.1 or 1 μg of FLAG-STAT2-FLAG, 0.1 μg NS2b-3, and either 1 μg E- clv NS5-HA or an empty vector plasmid. Cells were then lysed and examined by Western blotting using FLAG antibody and long-term film exposure to detect any additional low-intensity bands. The arrow indicates the expected size of FLAG-STAT2-FLAG. The asterisk marks a nonspecific band running at the same mobility of FLAG-STAT2-FLAG. (E) 293T cells were cotransfected with NS2b-3 and the plasmids indicated at the top. Twenty-four hours posttransfection, cells were sorted for GFP-positive cells by FACS, lysed, and examined via Western blotting using STAT2-, GFP-, HA-, FLAG-, and GAPDH-specific antibodies.
Figure Legend Snippet: Inhibitors of the ubiquitin-proteasome pathway prevent STAT2 degradation by DENV NS5. (A) wtVero or Vero cells stably expressing the DEN1 replicon were treated with the indicated amounts of MG132. Sixteen hours posttreatment, cells were lysed and examined for ubiquitin, STAT2, STAT1, NS5, and GAPDH levels via Western blotting. (B) STAT2-deficient U6A cells were transfected with HA-ubiquitin, STAT2-FLAG, STAT1-GFP, NS2b-3, and either E- clv NS5-HA or an empty vector plasmid. Cells were then treated with lactacystin for 8 h and subsequently lysed and examined by Western blotting using ubiquitin, STAT2, STAT1, HA, and tubulin antibodies. (C) 293T cells were cotransfected with NS2b-3-HA and the plasmids indicated at the top. Ten hours posttransfection, cells were treated with the indicated amounts of lactacystin. Twenty-four hours posttransfection, cells were sorted for GFP-positive cells by FACS, lysed, and examined by Western blotting using ubiquitin-, GFP-, HA-, and GAPDH-specific antibodies. (D) STAT2-deficient U6A cells were transfected with 0.1 or 1 μg of FLAG-STAT2-FLAG, 0.1 μg NS2b-3, and either 1 μg E- clv NS5-HA or an empty vector plasmid. Cells were then lysed and examined by Western blotting using FLAG antibody and long-term film exposure to detect any additional low-intensity bands. The arrow indicates the expected size of FLAG-STAT2-FLAG. The asterisk marks a nonspecific band running at the same mobility of FLAG-STAT2-FLAG. (E) 293T cells were cotransfected with NS2b-3 and the plasmids indicated at the top. Twenty-four hours posttransfection, cells were sorted for GFP-positive cells by FACS, lysed, and examined via Western blotting using STAT2-, GFP-, HA-, FLAG-, and GAPDH-specific antibodies.

Techniques Used: Stable Transfection, Expressing, Western Blot, Transfection, Plasmid Preparation, FACS

DENV NS5 interacts with STAT2. (A) 293T cells were cotransfected with plasmids expressing FLAG-tagged STAT1 (STAT1-FLAG) and STAT2 (STAT2-FLAG) and empty plasmid (empty) or plasmid expressing HA-tagged DENV NS5 (NS5-HA), DENV core (CORE-HA), or NiV-V (HA-NiV-V) proteins. Lysates were then immunoprecipitated with anti-HA antibody (IP HA), and Western blotting was performed using anti-HA and anti-FLAG antibodies. Asterisks mark the heavy and light chains from the HA antibody. (B) 293T cells were cotransfected with plasmids expressing NS5-HA and either STAT1-FLAG or STAT2-FLAG. Lysates were then immunoprecipitated with anti-FLAG antibody (IP FLAG), and Western blotting was performed using anti-HA and anti-FLAG antibodies. TCE, total cell extracts were subjected to Western blotting using anti-HA, anti-FLAG, and anti-GAPDH antibodies.
Figure Legend Snippet: DENV NS5 interacts with STAT2. (A) 293T cells were cotransfected with plasmids expressing FLAG-tagged STAT1 (STAT1-FLAG) and STAT2 (STAT2-FLAG) and empty plasmid (empty) or plasmid expressing HA-tagged DENV NS5 (NS5-HA), DENV core (CORE-HA), or NiV-V (HA-NiV-V) proteins. Lysates were then immunoprecipitated with anti-HA antibody (IP HA), and Western blotting was performed using anti-HA and anti-FLAG antibodies. Asterisks mark the heavy and light chains from the HA antibody. (B) 293T cells were cotransfected with plasmids expressing NS5-HA and either STAT1-FLAG or STAT2-FLAG. Lysates were then immunoprecipitated with anti-FLAG antibody (IP FLAG), and Western blotting was performed using anti-HA and anti-FLAG antibodies. TCE, total cell extracts were subjected to Western blotting using anti-HA, anti-FLAG, and anti-GAPDH antibodies.

Techniques Used: Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot

Expression of a precursor form of DENV NS5 cleaved by the DENV protease results in reduced STAT2 levels. (A) 293T cells were transfected with the indicated constructs. Twenty-four hours posttransfection, cells were sorted for GFP-positive cells by FACS, subsequently lysed, and examined via Western blotting using GFP-, HA-, STAT1-, STAT2-, and GAPDH-specific antibodies. Schematics of the transfected constructs are shown at the bottom. ORFs that contain an “sp” at the N terminus have a signal peptide which directs the entire polyprotein to the surface of the ER for translation. Black arrows and red arrows indicate cleavage sites for cellular and the DEN2 viral proteases, respectively. The DEN2 active protease is highlighted in red. Densitometry analysis of the levels of STAT2 and NS5 are included on the far right, and levels are calculated relative to the levels in lane 1, with a value of 1 in the case of NS5 levels being indicative of no detection (background levels). (B) Same as above (A). Mutation of the DENV protease recognition site at the N terminus of NS5 is indicated by the blue dashes. The DENV protease labeled in blue indicates a serine-to-alanine mutation within the catalytic site of the DENV protease.
Figure Legend Snippet: Expression of a precursor form of DENV NS5 cleaved by the DENV protease results in reduced STAT2 levels. (A) 293T cells were transfected with the indicated constructs. Twenty-four hours posttransfection, cells were sorted for GFP-positive cells by FACS, subsequently lysed, and examined via Western blotting using GFP-, HA-, STAT1-, STAT2-, and GAPDH-specific antibodies. Schematics of the transfected constructs are shown at the bottom. ORFs that contain an “sp” at the N terminus have a signal peptide which directs the entire polyprotein to the surface of the ER for translation. Black arrows and red arrows indicate cleavage sites for cellular and the DEN2 viral proteases, respectively. The DEN2 active protease is highlighted in red. Densitometry analysis of the levels of STAT2 and NS5 are included on the far right, and levels are calculated relative to the levels in lane 1, with a value of 1 in the case of NS5 levels being indicative of no detection (background levels). (B) Same as above (A). Mutation of the DENV protease recognition site at the N terminus of NS5 is indicated by the blue dashes. The DENV protease labeled in blue indicates a serine-to-alanine mutation within the catalytic site of the DENV protease.

Techniques Used: Expressing, Transfection, Construct, FACS, Western Blot, Mutagenesis, Labeling

Cleaved NS5 is sufficient for reduced STAT2 levels and does not require an N-terminal glycine. (A) 293T cells were transfected with the indicated constructs. Twenty-four hours posttransfection, cells were sorted for GFP-positive cells by FACS, subsequently lysed, subjected to 4-to-20% SDS-polyacrylamide gel electrophoresis, and examined via Western blotting using GFP-, HA-, STAT1-, STAT2-, and GAPDH-specific antibodies. The TEV protease and its cleavage site are indicated by the green text and arrow, respectively. Cleavage sequences targeted by endogenous deubiquitinases are noted by a yellow arrow. Densitometry analyses of the levels of STAT2 and NS5 are included at the bottom, and levels were calculated relative to the levels in lane 1, with a value of 1 in the case of NS5 levels being indicative of no detection (background levels). (B) Same as above (A) except that lysates were run on a 7.5% SDS-polyacrylamide gel and subsequently analyzed by Western blotting using GFP-, STAT2-, and tubulin-specific antibodies.
Figure Legend Snippet: Cleaved NS5 is sufficient for reduced STAT2 levels and does not require an N-terminal glycine. (A) 293T cells were transfected with the indicated constructs. Twenty-four hours posttransfection, cells were sorted for GFP-positive cells by FACS, subsequently lysed, subjected to 4-to-20% SDS-polyacrylamide gel electrophoresis, and examined via Western blotting using GFP-, HA-, STAT1-, STAT2-, and GAPDH-specific antibodies. The TEV protease and its cleavage site are indicated by the green text and arrow, respectively. Cleavage sequences targeted by endogenous deubiquitinases are noted by a yellow arrow. Densitometry analyses of the levels of STAT2 and NS5 are included at the bottom, and levels were calculated relative to the levels in lane 1, with a value of 1 in the case of NS5 levels being indicative of no detection (background levels). (B) Same as above (A) except that lysates were run on a 7.5% SDS-polyacrylamide gel and subsequently analyzed by Western blotting using GFP-, STAT2-, and tubulin-specific antibodies.

Techniques Used: Transfection, Construct, FACS, Polyacrylamide Gel Electrophoresis, Western Blot

Related Articles

Staining:

Article Title: Decreased STAT5 phosphorylation and GATA-3 expression in NOX2 deficient T cells: Role in T helper development
Article Snippet: .. Phosphorylated STAT levels were determined by intracellular staining with antibodies specific for pY701-STAT1 (4a), pY705-STAT3 (4/P-STAT3), pY693-STAT4 (38/p-Stat4), pY694-STAT5 (47), and pY641-STAT6 (J71-773.58.11) (all BD Pharmingen). .. Total STAT5 levels were determined by staining with a purified pan-STAT5 antibody (89, BD Pharmingen) followed by a F(ab’)2 donkey anti-mouse secondary antibody conjugate (Jackson ImmunoResearch).

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    Becton Dickinson stat1
    HMPV inhibits <t>STAT1</t> phosphorylation of Vero cells only in infected cells. (A) Vero cells were infected for 24 h with HMPV at MOI of 1 in 6-well plates. Cells were lysed with RIPA buffer, run on 4-15% bis-tris gel, transferred to nitrocellulose membrane,
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    HMPV inhibits STAT1 phosphorylation of Vero cells only in infected cells. (A) Vero cells were infected for 24 h with HMPV at MOI of 1 in 6-well plates. Cells were lysed with RIPA buffer, run on 4-15% bis-tris gel, transferred to nitrocellulose membrane,

    Journal: Virology

    Article Title: Human metapneumovirus small hydrophobic (SH) protein downregulates type I IFN pathway signaling by affecting STAT1 expression and phosphorylation

    doi: 10.1016/j.virol.2016.04.022

    Figure Lengend Snippet: HMPV inhibits STAT1 phosphorylation of Vero cells only in infected cells. (A) Vero cells were infected for 24 h with HMPV at MOI of 1 in 6-well plates. Cells were lysed with RIPA buffer, run on 4-15% bis-tris gel, transferred to nitrocellulose membrane,

    Article Snippet: To determine the effect of HMPV infection on STAT1, Vero cells were infected at an MOI of 1, treated with IFNα 24 hours later, then analyzed for STAT1 expression and phosphorylation by western blot ( ).

    Techniques: Infection

    Type I IFN secreted by infected BEAS-2B cells inhibits STAT1 phosphorylation in neighboring uninfected cells. (A) BEAS-2B cells were infected with HMPV for 24 h at an MOI of 1 in 6-well plates, then stained with polyclonal anti-HMPV antibody and gated

    Journal: Virology

    Article Title: Human metapneumovirus small hydrophobic (SH) protein downregulates type I IFN pathway signaling by affecting STAT1 expression and phosphorylation

    doi: 10.1016/j.virol.2016.04.022

    Figure Lengend Snippet: Type I IFN secreted by infected BEAS-2B cells inhibits STAT1 phosphorylation in neighboring uninfected cells. (A) BEAS-2B cells were infected with HMPV for 24 h at an MOI of 1 in 6-well plates, then stained with polyclonal anti-HMPV antibody and gated

    Article Snippet: To determine the effect of HMPV infection on STAT1, Vero cells were infected at an MOI of 1, treated with IFNα 24 hours later, then analyzed for STAT1 expression and phosphorylation by western blot ( ).

    Techniques: Infection, Staining

    HMPV inhibits STAT1 phosphorylation in BEAS-2B cells. (A) BEAS-2B cells were infected for 24 h with HMPV at an MOI of 1 in 6-well plates. Cells were lysed with RIPA buffer, run on 4-15% bis-tris gel, transferred to nitrocellulose membrane, and probed

    Journal: Virology

    Article Title: Human metapneumovirus small hydrophobic (SH) protein downregulates type I IFN pathway signaling by affecting STAT1 expression and phosphorylation

    doi: 10.1016/j.virol.2016.04.022

    Figure Lengend Snippet: HMPV inhibits STAT1 phosphorylation in BEAS-2B cells. (A) BEAS-2B cells were infected for 24 h with HMPV at an MOI of 1 in 6-well plates. Cells were lysed with RIPA buffer, run on 4-15% bis-tris gel, transferred to nitrocellulose membrane, and probed

    Article Snippet: To determine the effect of HMPV infection on STAT1, Vero cells were infected at an MOI of 1, treated with IFNα 24 hours later, then analyzed for STAT1 expression and phosphorylation by western blot ( ).

    Techniques: Infection

    Transient expression of HMPV SH protein inhibits STAT1 phosphorylation. (A) Vero cells transfected with HMPV GFP-fusion proteins were analyzed compared to untransfected cells in the same culture using flow cytometry. (B) Transfected cells were analyzed

    Journal: Virology

    Article Title: Human metapneumovirus small hydrophobic (SH) protein downregulates type I IFN pathway signaling by affecting STAT1 expression and phosphorylation

    doi: 10.1016/j.virol.2016.04.022

    Figure Lengend Snippet: Transient expression of HMPV SH protein inhibits STAT1 phosphorylation. (A) Vero cells transfected with HMPV GFP-fusion proteins were analyzed compared to untransfected cells in the same culture using flow cytometry. (B) Transfected cells were analyzed

    Article Snippet: To determine the effect of HMPV infection on STAT1, Vero cells were infected at an MOI of 1, treated with IFNα 24 hours later, then analyzed for STAT1 expression and phosphorylation by western blot ( ).

    Techniques: Expressing, Transfection, Flow Cytometry, Cytometry