Journal:

Article Title: Signalling events involved in interferon-?-inducible macrophage nitric oxide generation

doi: 10.1046/j.1365-2567.2003.01620.x

Figure Lengend Snippet: Nuclear translocation of signal transducer and activator of transcription 1α (STAT1α) and nuclear factor-κB (NF-κB) transcription factors in interferon-γ (IFN-γ)-stimulated J774 macrophages. Nuclear extracts from cells either left untreated or stimulated with IFN-γ (100 U/ml) for different time-periods (0–4 hr) were incubated with a [γ-32P]-labelled STAT1α (consensus sequence) (a), NF-κB (b), or STAT1α [IFN-γ-activated site (GAS)/inducible nitric oxide synthase (iNOS) sequence] (c) probe and were subjected to electrophoretic mobility shift assay (EMSA) analysis. The last lane in each panel represents nuclear extracts from IFN-γ-treated cells incubated with an excess of unlabelled oligonucleotide in order to evaluate band specificity. (d) For supershift assays, nuclear extracts from cells stimulated with IFN-γ (1 hr) were or were not incubated with a specific antibody against STAT1α for 1 hr before EMSA. Binding specificity was tested by adding, to nuclear extracts from 1-hr IFN-γ-treated cells, a 100-fold molar excess of either a cold GAS/iNOS oligonucleotide or a non-specific Oct-2A probe. (e) Macrophages were stimulated with IFN-γ (100 U/ml) for different time-periods (0–4 hr). Following RNA extraction, STAT1α and IκBα gene expression were determined using Northern blot analysis. Equal RNA loading was confirmed by hybridization with a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) cDNA probe. These results are representative of one of three separate experiments.

Article Snippet: Oligonucleotides specific for STAT1 (consensus sequence) and NF-κB (consensus sequence) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Translocation Assay, Incubation, Sequencing, Electrophoretic Mobility Shift Assay, Binding Assay, RNA Extraction, Gene Expression, Northern Blot, Hybridization

Journal:

Article Title: Signalling events involved in interferon-?-inducible macrophage nitric oxide generation

doi: 10.1046/j.1365-2567.2003.01620.x

Figure Lengend Snippet: Effect of specific second-messenger inhibitors on interferon-γ (IFN-γ)-induced signal transducer and activator of transcription 1α (STAT1α) binding to the murine inducible nitric oxide synthase (iNOS) promoter. J774 macrophages were treated with specific inhibitors against extracellular signal-regulated kinase 1 and 2 (Erk1/Erk2) (50 µm of apigenin, lanes 3 and 4), Janus kinase 2 (JAK2) (75 µm of AG-490, lanes 5 and 6) and MEK1/2 (50 µm of PD 98059, lanes 7 and 8) for 1 hr prior to IFN-γ stimulation for 15 min. The last lane represents nuclear extracts preincubated with unlabelled oligonucleotide (100 ×) for band specificity evaluation. This result is representative of one of three different experiments.

Article Snippet: Oligonucleotides specific for STAT1 (consensus sequence) and NF-κB (consensus sequence) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Binding Assay

Journal:

Article Title: Signalling events involved in interferon-?-inducible macrophage nitric oxide generation

doi: 10.1046/j.1365-2567.2003.01620.x

Figure Lengend Snippet: Time-dependent activation of Janus kinase 2 (JAK2), signal transducer and activator of transcription 1α (STAT1α) and extracellular signal-regulated kinase 1 and 2 (Erk1/Erk2) by interferon-γ (IFN-γ) in J774 macrophages: effect of signalling inhibitors on their phosphorylation states. Cells were treated with IFN-γ (100 U/ml) for different time-periods (0–60 min) and cell lysates were subjected to immunoblot analysis by using antibodies specific for the phosphorylated forms of Janus kinase 2 (JAK2) (a); STAT1α (b) and (c); and Erk1/Erk2 (d). (e) To monitor IFN-γ-dependent Erk1/Erk2, JAK2 and STAT1α activation in the presence of apigenin, AG-490 or PD 98059, cells were treated with maximal doses of these specific inhibitors for 1 hr prior to stimulation with IFN-γ (for 15 min). Cell lysates were then subjected to Western blotting and incubated with the same antibodies mentioned above (for panels a–d). Equal protein loading was verified by using anti-JAK2, anti-STAT1α and anti-Erk1/Erk2 specific antibodies. Results are representative of three experiments performed independently.

Article Snippet: Oligonucleotides specific for STAT1 (consensus sequence) and NF-κB (consensus sequence) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Activation Assay, Phospho-proteomics, Western Blot, Incubation

Journal: Redox Biology

Article Title: Zinc deficiency affects the STAT1/3 signaling pathways in part through redox-mediated mechanisms

doi: 10.1016/j.redox.2016.12.027

Figure Lengend Snippet: Gestational MZD affects phosphorylations of STAT1 and STAT3 in E19 brain total fractions. From gestation day 0 through 19, dams were fed ad libitum control (C) or MZD diets. STAT1 and STAT3 contents and phosphorylations in E19 brain total homogenates were measured by Western blot. (A) Representative images for pY 701 -STAT1, pS 727 -STAT1, STAT1, and β-actin (top); and band quantifications expressed as pY 701 -STAT1/STAT1, pS 727 -STAT1/STAT1 and total STAT1/β-actin (bottom). (B) Representative images for pY 705 -STAT3, pS 727 -STAT3, STAT3, and β-actin (top), and band quantifications expressed as pY 705 -STAT3/STAT3, pS 727 -STAT3/STAT3 and STAT3/β-actin (bottom). Results were normalized to control values and are shown as means±S.E.M. of 6 animals per group. *, ** Significantly different compared to C groups ( P <0.05 and P <0.01, respectively, one-way ANOVA).

Article Snippet: Antibodies for STAT1, STAT3, β-actin, α-tubulin, β-tubulin and heterogeneous nuclear ribonucleoprotein A1 (hnRNP), and the oligonucleotides containing the consensus sequence for STAT1 and STAT3 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Western Blot

Journal: Redox Biology

Article Title: Zinc deficiency affects the STAT1/3 signaling pathways in part through redox-mediated mechanisms

doi: 10.1016/j.redox.2016.12.027

Figure Lengend Snippet: Gestational MZD affects DNA binding and content of STAT1 and STAT3 in nuclear fractions isolated from E19 brains. From gestation day 0 through 19, dams were fed ad libitum control (C) or MZD diets. Nuclear and cytosolic fractions were prepared from E19 brains as described in the Materials and Methods section. (A) EMSA for STAT1 and STAT3 in nuclear and cytosolic fractions. To determine the specificity of each transcription factor-DNA complex, a control nuclear fraction was incubated in the presence of a 100-fold molar excess of unlabeled oligonucleotide containing the consensus sequence for either the specific (S) or an unspecific (U) transcription factor before the binding assay. Bands were quantified and the ratio nuclear/cytosolic DNA binding (NF/CF) was calculated. (B) Western blots for pY 701 -STAT1, pS 727 -STAT1, and STAT1 (left); and pY 705 -STAT3, pS 727 -STAT3, and STAT3 (right); and hnRNP as the nuclear housekeeping protein. After quantification, results were expressed as pY 701 -STAT1/STAT1, pS 727 -STAT1/STAT1 and STAT1/hnRNP (bottom left), or pY 705 -STAT3/STAT3, pS 727 -STAT3/STAT3, and STAT3/hnRNP (bottom right). Results were normalized to control values and are shown as means±S.E.M. of 6 animals per group. *, ** Significantly different compared to C groups ( P <0.05 and P <0.01, respectively, one-way ANOVA).

Article Snippet: Antibodies for STAT1, STAT3, β-actin, α-tubulin, β-tubulin and heterogeneous nuclear ribonucleoprotein A1 (hnRNP), and the oligonucleotides containing the consensus sequence for STAT1 and STAT3 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Binding Assay, Isolation, Incubation, Sequencing, Western Blot

Journal: Redox Biology

Article Title: Zinc deficiency affects the STAT1/3 signaling pathways in part through redox-mediated mechanisms

doi: 10.1016/j.redox.2016.12.027

Figure Lengend Snippet: Zinc deficiency affects total protein content and phosphorylation patterns of STAT1 and STAT3 in IMR-32 total fractions. Total cell fractions were isolated from IMR-32 cells incubated for 24 h in control (C) or zinc -depleted (1.5 μM zinc (1.5Zn)) medium. STAT1 and STAT3 contents and phosphorylations were measured by Western blot. (A) Representative images for pY 701 -STAT1, pS 727 -STAT1, STAT1, and β-tubulin as the housekeeping protein (top), and their quantification expressed as pY 701 -STAT1/STAT1, pS 727 -STAT1/STAT1 and STAT1/β-tubulin (bottom). (B) Representative images for pY 705 -STAT3, pS 727 -STAT3, STAT3, and β-tubulin (top), and their quantification expressed as pY 705 -STAT3/STAT3, pS 727 -STAT3/STAT3, and STAT3/β-tubulin (bottom). Results were normalized to control values (dotted lined) and shown as means±S.E.M. of four independent experiments. *, ** Significantly different compared to C groups ( P <0.05 and P <0.01, respectively, one-way ANOVA).

Article Snippet: Antibodies for STAT1, STAT3, β-actin, α-tubulin, β-tubulin and heterogeneous nuclear ribonucleoprotein A1 (hnRNP), and the oligonucleotides containing the consensus sequence for STAT1 and STAT3 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Isolation, Incubation, Western Blot

Journal: Redox Biology

Article Title: Zinc deficiency affects the STAT1/3 signaling pathways in part through redox-mediated mechanisms

doi: 10.1016/j.redox.2016.12.027

Figure Lengend Snippet: Zinc deficiency affects nuclear STAT1- and STAT3-DNA binding, and transactivation of STAT1 and STAT3-dependent genes in IMR-32 cells . Nuclear and total cell fractions were isolated after incubating IMR-32 cells for 24 h in control medium (C), or in chelated medium containing 1.5 μM zinc (1.5Zn), or 15 μM zinc (15Zn). (A) Representative image of an EMSA for STAT1 and STAT3 in nuclear fractions (top). A control nuclear fraction was incubated in the presence of a 100-fold molar excess of unlabeled oligonucleotide containing the consensus sequence for aspecific (S) transcription factor before the binding assay. Bands were quantified, results expressed as the ratio nuclear/total fraction binding (NF/TF) for STAT1 (white bars) and STAT3 (grey bars), and normalized to control values. Results are shown as means±S.E.M. of three independent experiments. (B) Transactivation of STAT1- and STAT3-driven luciferase was measured as described in Materials and Methods in cells incubated for 24 h in control, 1.5Zn or 15Zn medium. Data are expressed as the ratio luciferase activity/β-galactosidase activity. Results are shown as means±S.E.M of three independent experiments. *Significantly different compared to the C groups ( P <0.05, one-way ANOVA).

Article Snippet: Antibodies for STAT1, STAT3, β-actin, α-tubulin, β-tubulin and heterogeneous nuclear ribonucleoprotein A1 (hnRNP), and the oligonucleotides containing the consensus sequence for STAT1 and STAT3 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Binding Assay, Isolation, Incubation, Sequencing, Luciferase, Activity Assay

Journal: Redox Biology

Article Title: Zinc deficiency affects the STAT1/3 signaling pathways in part through redox-mediated mechanisms

doi: 10.1016/j.redox.2016.12.027

Figure Lengend Snippet: Zinc deficiency affects total protein content and phosphorylation patterns of STAT1 and STAT3 in IMR-32 cell nuclear fractions. Nuclear fractions were isolated from IMR-32 cells incubated for 24 h in control (C) or 1.5Zn medium. STAT1 and STAT3 content and phosphorylations were measured by Western blot. (A) Representative images for pY 701 -STAT1, pS 727 -STAT1, STAT1, and hnRNP as the nuclear housekeeping protein (top), and their quantification expressed as pY 701 -STAT1/STAT1, pS 727 -STAT1/STAT1 and STAT1/hnRNP (bottom). (B) Representative images for pY 705 -STAT3, pS 727 -STAT3, STAT3, and hnRNP (top), and their quantification expressed as pY 705 -STAT3/STAT3, pS 727 -STAT3/STAT3, and STAT3/hnRNP (bottom). Results were normalized to control values (dotted lined) and shown as means±S.E.M. of four independent experiments. *, ** Significantly different compared to C groups ( P <0.05 and P <0.01, respectively, one-way ANOVA).

Article Snippet: Antibodies for STAT1, STAT3, β-actin, α-tubulin, β-tubulin and heterogeneous nuclear ribonucleoprotein A1 (hnRNP), and the oligonucleotides containing the consensus sequence for STAT1 and STAT3 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Isolation, Incubation, Western Blot

Journal: Redox Biology

Article Title: Zinc deficiency affects the STAT1/3 signaling pathways in part through redox-mediated mechanisms

doi: 10.1016/j.redox.2016.12.027

Figure Lengend Snippet: α-Lipoic acid prevents zinc deficiency-induced impaired STAT1 and STAT3 nuclear import. Nuclear fraction and cytosolic fraction were isolated from IMR-32 cells incubated for 24 h in control (C), or zinc depleted (1.5Zn) medium with or without supplementation with 0.5mM α-lipoic acid (LA). Western blots for: (A) STAT1 and hnRNP as housekeeping protein in the nuclear fraction (top left), and STAT1 and β-actin as housekeeping protein in the cytosolic fraction (top right), and band quantification expressed as the ratio of total STAT1 nuclear/cytosolic (bottom); (B) STAT3 and hnRNP in the nuclear fraction (top left) and STAT3 and β-actin in the cytosolic fraction (top right), and band quantification expressed as the ratio total STAT3 nuclear/cytosolic fractions (bottom). Results were normalized to controls values, and shown as means±S.E.M. of three independent experiments. *, ** Significantly different compared to C groups ( P <0.05 and P <0.01, respectively, one-way ANOVA).

Article Snippet: Antibodies for STAT1, STAT3, β-actin, α-tubulin, β-tubulin and heterogeneous nuclear ribonucleoprotein A1 (hnRNP), and the oligonucleotides containing the consensus sequence for STAT1 and STAT3 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Isolation, Incubation, Western Blot

Journal: Redox Biology

Article Title: Zinc deficiency affects the STAT1/3 signaling pathways in part through redox-mediated mechanisms

doi: 10.1016/j.redox.2016.12.027

Figure Lengend Snippet: STAT1 and STAT3 require a functional cytoskeleton for nuclear translocation in IMR-32 cells. Nuclear fractions and total fractions were prepared from IMR-32 cells incubated for 24 h in control medium (C) with or without the addition of 0.5 μM vinblastine (Vb), 0.5 μM colchicine (Col), or 0.5 μM cytochalasin D (Cyt D). (A) EMSA for STAT1 in nuclear and total fractions (top), and band quantifications (bottom). (B) EMSA for STAT3 in nuclear and total fractions (top), and band quantifications (bottom). Results were expressed as the ratio nuclear/total fractions of DNA binding (NF/TF) and normalized to their control levels. Results are shown as means±S.E.M. of three independent experiments. *Significantly different compared to C without inhibitors ( P <0.05, one-way ANOVA).

Article Snippet: Antibodies for STAT1, STAT3, β-actin, α-tubulin, β-tubulin and heterogeneous nuclear ribonucleoprotein A1 (hnRNP), and the oligonucleotides containing the consensus sequence for STAT1 and STAT3 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Functional Assay, Translocation Assay, Incubation, Binding Assay

Journal: Redox Biology

Article Title: Zinc deficiency affects the STAT1/3 signaling pathways in part through redox-mediated mechanisms

doi: 10.1016/j.redox.2016.12.027

Figure Lengend Snippet: α-Lipoic acid differentially affects STAT1 and STAT3 phosphorylation in zinc deficient IMR-32 cells. Total fractions were prepared from IMR-32 cells incubated for 24 h in control (C), or zinc deficient (1.5Zn) medium with or without supplementation with 0.5mM α-lipoic acid (LA). STAT1 and STAT3 content and phosphorylation were measured by Western blot. (A) Representative images for pY 701 -STAT1, pS 727 -STAT1, STAT1, and β-actin and their quantification expressed as pY 701 -STAT1/STAT1, pS 727 -STAT1/STAT1, and STAT1/β-actin (bottom). (B) Representative images for pY 705 -STAT3, STAT3 and β-actin, and their quantification expressed as pY 705 -STAT3/STAT3, and STAT3/β-actin. Results were normalized to control values and shown as means±S.E.M. of four independent experiments. *, **, *** Significantly different compared to C groups ( P <0.05, P <0.01, and P <0.001, respectively, one-way ANOVA).

Article Snippet: Antibodies for STAT1, STAT3, β-actin, α-tubulin, β-tubulin and heterogeneous nuclear ribonucleoprotein A1 (hnRNP), and the oligonucleotides containing the consensus sequence for STAT1 and STAT3 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Incubation, Western Blot