rabbit polyclonal igg 9172 against total human stat1  (Cell Signaling Technology Inc)

Journal: Respiratory Research

Article Title: Inhibition of IFN-γ-dependent antiviral airway epithelial defense by cigarette smoke

doi: 10.1186/1465-9921-11-64

Figure Lengend Snippet: Cigarette smoke extract decreases type II interferon-induced protein expression . A : ICAM-1 cell surface protein levels were determined using an enzyme-linked immunoassay with hTBE cell monolayers that were first treated with media without or with CSE at the indicated concentration for 4 hours. Cells were then incubated for 20 hours in media containing the same CSE concentration without or with IFN-γ. Values are expressed as mean ± S.D. ( n = 3 replicates), and a significant difference ( p < 0.05) in IFN-γ-induced levels between cells treated versus not treated with CSE is indicated by an asterisk . B : IRF-9, ICAM-1, HSP90, and β-actin protein levels were assessed using immunoblot analysis of extracts from hTBE cells that were first treated with media without or with CSE at the indicated concentration for 4 hours, followed by incubation for 8 hours in media containing the same CSE concentration without or with IFN-γ. C : Total Stat1 and β-actin protein levels were assessed using immunoblot analysis of extracts from hTBE cells that were first treated with media without or with CSE at the indicated concentration for 4 hours, followed by incubation for 8 hours in media containing the same CSE concentration without or with IFN-γ. In B and C , protein levels were quantified using band densitometry of immunoblot analyses from separate experiments with the value for cells not exposed to CSE but treated with IFN-γ set at 1 in each experiment. Values are expressed as mean ± S.D. ( n = 4 experiments) in the bar graph adjacent to a representative immunoblot analysis, and a significant difference ( p < 0.05) in IFN-γ-induced levels between cells treated versus not treated with CSE is indicated by an asterisk .

Article Snippet: Primary antibodies used to detect specific cellular and nuclear proteins were: mouse IgG1 mAb clone 6 against human interferon regulatory factor-9 (IRF-9) from BD Transduction Laboratories (Lexington, KY); rabbit polyclonal IgG 4915 against human ICAM-1, rabbit polyclonal IgG 9172 against total human Stat1, and rabbit polyclonal IgG 9171 against tyrosine-701 phosphorylated human Stat1 from Cell Signaling Technology (Beverly, MA); rabbit polyclonal antiserum against human heat shock protein (HSP)-90 from Assay Designs (Ann Arbor, MI); mouse IgG2a mAb clone AC-74 against human β-actin from Sigma-Aldrich (St. Louis, MO); rabbit polyclonal IgG ab4742 against serine-727 phosphorylated human Stat1 from Abcam (Cambridge, MA); goat polyclonal IgG against human RSV proteins from Biodesign International (Saco, ME).

Techniques: Expressing, Concentration Assay, Incubation, Western Blot

Journal: Respiratory Research

Article Title: Inhibition of IFN-γ-dependent antiviral airway epithelial defense by cigarette smoke

doi: 10.1186/1465-9921-11-64

Figure Lengend Snippet: Cigarette smoke extract inhibits type II interferon-induced Stat1 activation . A : Tyrosine-701 and serine-727 phosphorylated and total Stat1, and β-actin levels were assessed using immunoblot analysis of extracts from hTBE cells that were first treated with media without or with CSE at the indicated concentrations for 4 hours, followed by incubation for 30 minutes in media containing the same CSE concentration without or with IFN-γ. B : Tyrosine-701 and serine-727 phosphorylated and total Stat1, ICAM-1, and β-actin levels were assessed using immunoblot analysis of extracts from hTBE cells that were first treated with media without or with CSE at the indicated concentrations for 4 hours, followed by incubation for 20 hours in media containing the same CSE concentration without or with IFN-γ. In A and B , protein levels were quantified using band densitometry of immunoblot analyses from separate experiments with the value for cells not exposed to CSE but treated with IFN-γ set at 1 in each experiment. Values are expressed as mean ± S.D. ( n = 3 experiments) in the bar graph adjacent to a representative immunoblot analysis, and a significant difference ( p < 0.05) in IFN-γ-induced levels between cells treated versus not treated with CSE is indicated by an asterisk .

Article Snippet: Primary antibodies used to detect specific cellular and nuclear proteins were: mouse IgG1 mAb clone 6 against human interferon regulatory factor-9 (IRF-9) from BD Transduction Laboratories (Lexington, KY); rabbit polyclonal IgG 4915 against human ICAM-1, rabbit polyclonal IgG 9172 against total human Stat1, and rabbit polyclonal IgG 9171 against tyrosine-701 phosphorylated human Stat1 from Cell Signaling Technology (Beverly, MA); rabbit polyclonal antiserum against human heat shock protein (HSP)-90 from Assay Designs (Ann Arbor, MI); mouse IgG2a mAb clone AC-74 against human β-actin from Sigma-Aldrich (St. Louis, MO); rabbit polyclonal IgG ab4742 against serine-727 phosphorylated human Stat1 from Abcam (Cambridge, MA); goat polyclonal IgG against human RSV proteins from Biodesign International (Saco, ME).

Techniques: Activation Assay, Western Blot, Incubation, Concentration Assay

Journal: Respiratory Research

Article Title: Inhibition of IFN-γ-dependent antiviral airway epithelial defense by cigarette smoke

doi: 10.1186/1465-9921-11-64

Figure Lengend Snippet: Cigarette smoke extract has a delayed effect on epithelial cell Stat1 activation . A : Tyrosine-701 phosphorylated and total Stat1, and β-actin levels were assessed using immunoblot analysis of extracts from hTBE cells that were first treated with media without or with CSE at the indicated concentrations for 4 hours, followed by incubation for 8 hours in media containing the same CSE concentration without or with IFN-γ. Protein levels were quantified using band densitometry of immunoblot analyses from separate experiments with the value for cells not exposed to CSE but treated with IFN-γ set at 1 in each experiment. Values are expressed as mean ± S.D. ( n = 3 experiments) in the bar graph adjacent to a representative immunoblot analysis, and a significant difference ( p < 0.05) in IFN-γ-induced levels between cells treated versus not treated with CSE is indicated by an asterisk . B : Tyrosine-701 phosphorylated and total Stat1, and β-actin levels were assessed using immunoblot analysis of extracts from hTBE cells that were first treated with media without or with CSE at the indicated concentrations for 12 hours, followed by incubation for 30 minutes in media containing the same CSE concentration without or with IFN-γ. C : Tyrosine-701 phosphorylated and total Stat1, and β-actin levels were assessed using immunoblot analysis of extracts from hTBE cells that were first treated with media without or with CSE at the indicated concentrations for 12 hours, followed by incubation for 30 minutes in media without or with IFN-γ that had been preincubated without or with the same CSE concentration alone for 8 hours.

Article Snippet: Primary antibodies used to detect specific cellular and nuclear proteins were: mouse IgG1 mAb clone 6 against human interferon regulatory factor-9 (IRF-9) from BD Transduction Laboratories (Lexington, KY); rabbit polyclonal IgG 4915 against human ICAM-1, rabbit polyclonal IgG 9172 against total human Stat1, and rabbit polyclonal IgG 9171 against tyrosine-701 phosphorylated human Stat1 from Cell Signaling Technology (Beverly, MA); rabbit polyclonal antiserum against human heat shock protein (HSP)-90 from Assay Designs (Ann Arbor, MI); mouse IgG2a mAb clone AC-74 against human β-actin from Sigma-Aldrich (St. Louis, MO); rabbit polyclonal IgG ab4742 against serine-727 phosphorylated human Stat1 from Abcam (Cambridge, MA); goat polyclonal IgG against human RSV proteins from Biodesign International (Saco, ME).

Techniques: Activation Assay, Western Blot, Incubation, Concentration Assay

Journal: Respiratory Research

Article Title: Inhibition of IFN-γ-dependent antiviral airway epithelial defense by cigarette smoke

doi: 10.1186/1465-9921-11-64

Figure Lengend Snippet: N-acetylcysteine inhibits cigarette smoke effects on type II interferon-induced responses . A : ICAM-1 cell surface protein levels were determined using an enzyme-linked immunoassay with hTBE cells that were first treated in media without or with NAC for 1 hour. Cells were then incubated in media containing the antioxidant without or with CSE at the indicated concentrations for 4 hours, followed by incubation for 20 hours with the same compounds without or with IFN-γ. Values are expressed as mean ± S.D. ( n = 3 replicates) and a significant difference ( p < 0.05) in comparable CSE-treated levels between cells not incubated versus incubated with NAC is indicated by an asterisk . B : Tyrosine-701 phosphorylated and total Stat1, ICAM-1, and β-actin protein levels were assessed using immunoblot analysis of extracts from hTBE cells that were first treated in media without or with NAC for 1 hour. Cells were then incubated in media containing the antioxidant without or with CSE at the indicated concentrations for 4 hours, followed by incubation for 8 hours with the same compounds without or with IFN-γ.

Article Snippet: Primary antibodies used to detect specific cellular and nuclear proteins were: mouse IgG1 mAb clone 6 against human interferon regulatory factor-9 (IRF-9) from BD Transduction Laboratories (Lexington, KY); rabbit polyclonal IgG 4915 against human ICAM-1, rabbit polyclonal IgG 9172 against total human Stat1, and rabbit polyclonal IgG 9171 against tyrosine-701 phosphorylated human Stat1 from Cell Signaling Technology (Beverly, MA); rabbit polyclonal antiserum against human heat shock protein (HSP)-90 from Assay Designs (Ann Arbor, MI); mouse IgG2a mAb clone AC-74 against human β-actin from Sigma-Aldrich (St. Louis, MO); rabbit polyclonal IgG ab4742 against serine-727 phosphorylated human Stat1 from Abcam (Cambridge, MA); goat polyclonal IgG against human RSV proteins from Biodesign International (Saco, ME).

Techniques: Incubation, Western Blot

Journal: Respiratory Research

Article Title: Inhibition of IFN-γ-dependent antiviral airway epithelial defense by cigarette smoke

doi: 10.1186/1465-9921-11-64

Figure Lengend Snippet: GSH monoethyl ester inhibits cigarette smoke effects on type II interferon-induced responses . A : ICAM-1 cell surface protein levels were determined using an enzyme-linked immunoassay with hTBE cells that were first treated in media without or with GSH-MEE for 2 hours. Cells were then incubated in media containing the antioxidant without or with CSE at the indicated concentrations for 4 hours, followed by incubation for 20 hours with the same compounds without or with IFN-γ. Values are expressed as mean ± S.D. ( n = 3 replicates) and a significant difference ( p < 0.01) in comparable CSE-treated levels between cells not incubated versus incubated with GSH-MEE is indicated by an asterisk . B : Tyrosine-701 phosphorylated and total Stat1, ICAM-1, and β-actin protein levels were assessed using immunoblot analysis of extracts from hTBE cells that were first treated in media without or with GSH-MEE for 2 hours. Cells were then incubated in media containing the antioxidant without or with CSE at the indicated concentrations for 4 hours, followed by incubation for 8 hours with the same compounds without or with IFN-γ.

Article Snippet: Primary antibodies used to detect specific cellular and nuclear proteins were: mouse IgG1 mAb clone 6 against human interferon regulatory factor-9 (IRF-9) from BD Transduction Laboratories (Lexington, KY); rabbit polyclonal IgG 4915 against human ICAM-1, rabbit polyclonal IgG 9172 against total human Stat1, and rabbit polyclonal IgG 9171 against tyrosine-701 phosphorylated human Stat1 from Cell Signaling Technology (Beverly, MA); rabbit polyclonal antiserum against human heat shock protein (HSP)-90 from Assay Designs (Ann Arbor, MI); mouse IgG2a mAb clone AC-74 against human β-actin from Sigma-Aldrich (St. Louis, MO); rabbit polyclonal IgG ab4742 against serine-727 phosphorylated human Stat1 from Abcam (Cambridge, MA); goat polyclonal IgG against human RSV proteins from Biodesign International (Saco, ME).

Techniques: Incubation, Western Blot

Journal: Respiratory Research

Article Title: Inhibition of IFN-γ-dependent antiviral airway epithelial defense by cigarette smoke

doi: 10.1186/1465-9921-11-64

Figure Lengend Snippet: Model for cigarette smoke effects on type II interferon signal transduction . Cigarette smoke inhibits type II interferon-dependent gene expression by decreasing Stat1 phosphorylation. A portion of this effect is mediated by reactive oxygen species (ROS). Decreased antiviral gene expression decreases epithelial cell responses to IFN-γ that inhibit viral infection.

Article Snippet: Primary antibodies used to detect specific cellular and nuclear proteins were: mouse IgG1 mAb clone 6 against human interferon regulatory factor-9 (IRF-9) from BD Transduction Laboratories (Lexington, KY); rabbit polyclonal IgG 4915 against human ICAM-1, rabbit polyclonal IgG 9172 against total human Stat1, and rabbit polyclonal IgG 9171 against tyrosine-701 phosphorylated human Stat1 from Cell Signaling Technology (Beverly, MA); rabbit polyclonal antiserum against human heat shock protein (HSP)-90 from Assay Designs (Ann Arbor, MI); mouse IgG2a mAb clone AC-74 against human β-actin from Sigma-Aldrich (St. Louis, MO); rabbit polyclonal IgG ab4742 against serine-727 phosphorylated human Stat1 from Abcam (Cambridge, MA); goat polyclonal IgG against human RSV proteins from Biodesign International (Saco, ME).

Techniques: Transduction, Gene Expression, Phospho-proteomics, Infection

Journal: Respiratory Research

Article Title: Differential effects of cytokines and corticosteroids on Toll-like receptor 2 expression and activity in human airway epithelia

doi: 10.1186/1465-9921-10-96

Figure Lengend Snippet: Cytokine-induced Stat1 activation does not persist during subsequent TLR2 stimulation . A) Phosphorylated and total Stat1 cellular protein levels were assessed using immunoblot analysis of extracts from human airway epithelia that were treated without or with media containing TNF-α (100 ng/ml) and IFN-γ (100 ng/ml) for 30 min. B). Phosphorylated and total Stat1 cellular protein levels were assessed using immunoblot analysis of extracts from human airway epithelia that were first treated without or with media containing TNF-α (100 ng/ml) and IFN-γ (100 ng/ml) for 24 hours, followed by incubation without or with Pam3CSK4 (25 mg/ml) for 30 min.

Article Snippet: Primary antibodies used to detect specific cellular and nuclear proteins were: mouse IgG1 mAb clone L35A5 against human IκBα, rabbit polyclonal IgG 9171 against human Stat1 phosphorylated on tyrosine-701, rabbit polyclonal IgG 9172 against human total Stat1, rabbit IgG mAb clone 3D7 against human p38 MAP kinase phosphorylated on threonine-180 and tyrosine-182, rabbit IgG mAb clone 7D6 against human total p38 MAP kinase from Cell Signaling Technology (Beverly, MA); mouse IgG2α mAb clone AC-74 against human β-actin from Sigma-Aldrich (St. Louis, MO); rabbit polyclonal antiserum against human heat shock protein (HSP)-90 from Assay Designs (Ann Arbor, MI).

Techniques: Activation Assay, Western Blot, Incubation