Journal: Immunologic Research

Article Title: Expression of JAK3, STAT2, STAT4, and STAT6 in pemphigus vulgaris

doi: 10.1007/s12026-020-09122-y

Figure Lengend Snippet: Immunoexpression of JAK/STAT proteins in the epidermis, normal skin, × 400. a Immunoexpression of JAK3 in the epidermis, normal skin, 10.73 ± 3.36. b Immunoexpression of STAT2 in the epidermis, normal skin, 11.06 ± 5.34. c Immunoexpression of STAT4 in the epidermis, normal skin, 18.59 ± 3.01. d Immunoexpression of STAT6 in the epidermis, normal skin, 11.56 ± 2.84

Article Snippet: Endogenous peroxidase activity was blocked by 0.3% hydrogen peroxide in distilled water and then, sections were rinsed with Tris-buffered saline (TBS, Dako, Denmark), and incubated with primary rabbit polyclonal antibody against STAT2 (Santa Cruz Biotechnology Inc.), mouse monoclonal antibody against STAT4 (Santa Cruz Biotechnology, Inc.), and primary rabbit polyclonal antibody against STAT6 (Santa Cruz biotechnology Inc.) and incubated overnight with mouse monoclonal antibody against JAK3.

Techniques:

Journal: Immunologic Research

Article Title: Expression of JAK3, STAT2, STAT4, and STAT6 in pemphigus vulgaris

doi: 10.1007/s12026-020-09122-y

Figure Lengend Snippet: Immunoexpression of JAK/STAT proteins in the epidermis, pemphigus vulgaris, × 400. Immunoexpression of JAK3 in the epidermis: a perilesional skin 14.38 ± 3.61; b skin lesions 18.89 ± 4.67, p > 0.05. Immunoexpression of STAT2 in the epidermis: c perilesional skin 15.79 ± 2.06; d skin lesions 17.15 ± 2.81, non-significant. Immunoexpression of STAT4 in the epidermis: e perilesional skin 24.10 ± 3.40; f skin lesions 29.08 ± 4.38, p < 0.05. Immunoexpression of STAT6 in the epidermis: g perilesional skin 18.21 ± 3.49; h skin lesions 27.85 ± 4.68, p < 0.05

Article Snippet: Endogenous peroxidase activity was blocked by 0.3% hydrogen peroxide in distilled water and then, sections were rinsed with Tris-buffered saline (TBS, Dako, Denmark), and incubated with primary rabbit polyclonal antibody against STAT2 (Santa Cruz Biotechnology Inc.), mouse monoclonal antibody against STAT4 (Santa Cruz Biotechnology, Inc.), and primary rabbit polyclonal antibody against STAT6 (Santa Cruz biotechnology Inc.) and incubated overnight with mouse monoclonal antibody against JAK3.

Techniques:

Journal: Immunologic Research

Article Title: Expression of JAK3, STAT2, STAT4, and STAT6 in pemphigus vulgaris

doi: 10.1007/s12026-020-09122-y

Figure Lengend Snippet: Morphometric analysis of JAK3 (upper left), STAT2 (upper right), STAT4 (lower left), and STAT6 (lower right) immunoexpression in keratinocytes. The results of semiquantitative analysis are expressed as the mean ± standard deviation. Control – normal skin. PV-P – pemphigus vulgaris perilesional skin, PV-L – pemphigus vulgaris skin lesions. The level of significance is defined where p < 0.05. JAK3: C vs. PV-L ( p < 0.05); PV-P vs. PV-L ( p < 0.05); STAT2: C vs. PV-L ( p < 0.05); STAT4: C vs. PV-P (NS), C vs. PV-L (NS); STAT6: C vs. PV-P ( p < 0.05), C vs. PV-L ( p < 0.05), PV-P vs. PV-L ( p < 0.05)

Article Snippet: Endogenous peroxidase activity was blocked by 0.3% hydrogen peroxide in distilled water and then, sections were rinsed with Tris-buffered saline (TBS, Dako, Denmark), and incubated with primary rabbit polyclonal antibody against STAT2 (Santa Cruz Biotechnology Inc.), mouse monoclonal antibody against STAT4 (Santa Cruz Biotechnology, Inc.), and primary rabbit polyclonal antibody against STAT6 (Santa Cruz biotechnology Inc.) and incubated overnight with mouse monoclonal antibody against JAK3.

Techniques: Standard Deviation, Control

Journal: Journal of Biological Chemistry

Article Title: Functional Relevance of the Conserved DNA-binding Domain of STAT2

doi: 10.1074/jbc.m500426200

Figure Lengend Snippet: FIG. 2. IFN-inducible tyrosine phos- phorylation of STAT1 and intact and mutant STAT2 proteins. A, cells were either left untreated () or treated () for 15 min with 5 ng/ml IFN alfacon-1, as indicated. Protein lysates were resolved by SDS-PAGE and immunoblotted with an anti-phospho-STAT2 antibody. Mem- branes were stripped and re-probed with anti-STAT2 and anti--actin antibodies to control for loading. B, similar to A, but antibodies were specific for phosphoryl- ated and total forms of STAT1.

Article Snippet: Antibodies against phospho-STAT2 and phospho-STAT1 were obtained from Cell Signaling Technology (Beverly, MA).

Techniques: Mutagenesis, SDS Page, Control

Journal: Journal of Biological Chemistry

Article Title: Functional Relevance of the Conserved DNA-binding Domain of STAT2

doi: 10.1074/jbc.m500426200

Figure Lengend Snippet: FIG. 3. Cells expressing the VV-II STAT2 exhibit defective IFN-induced biological responses and GAS-mediated transcriptional activation. A, 104 cells were treated with the indicated doses of IFN alfacon-1 for 16 h, and then challenged with EMCV. 24 h later, virus-induced cytopathic effects were quantitated spectrophotometrically. Data are expressed as percent protection from the cytopathic effects of EMCV compared with untreated, uninfected controls. Mean value S.E. of three independent experiments is shown. B, 2 103 cells were treated with the indicated doses of IFN alfacon-1 for 72 h. Cell proliferation was assessed spectrophotometrically. Data are expressed as percent inhibition compared with untreated, control cells. Mean value S.E. of three independent experiments is shown. C, ISRE luciferase gene reporter constructs were co-transfected into cells with a construct carrying the -galactosidase gene. 48 h after transfection, cells were either left untreated or treated with 5 ng/ml IFN alfacon-1 for 6 h. Luciferase activity was measured and values were normalized for the -galactosidase activity of each sample to control for transfection efficiency. Data are expressed as -fold increase of luciferase activity in response to IFN over untreated samples. Values are mean S.E. of three independent experiments. D, similar to C, but cells were transfected with an 8GAS luciferase gene reporter construct.

Article Snippet: Antibodies against phospho-STAT2 and phospho-STAT1 were obtained from Cell Signaling Technology (Beverly, MA).

Techniques: Expressing, Activation Assay, Virus, Inhibition, Control, Luciferase, Construct, Transfection, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: Functional Relevance of the Conserved DNA-binding Domain of STAT2

doi: 10.1074/jbc.m500426200

Figure Lengend Snippet: FIG. 4. Chromatin DNA binding ac- tivity of IFN-inducible STAT2:1 het- erodimers is reduced in cells express- ing VV-II STAT2. A, nuclear extracts from cells either left untreated () or treated () for 15 min with 5 ng/ml IFN alfacon-1 were incubated with 32P-labeled ISRE element probe. Untreated and IFN- treated nuclear extracts from human fi- brosarcoma HT-1080 cells were used as a positive control. Complexes were resolved by native gel electrophoresis and visual- ized by autoradiography. B, similar to A, but extracts were incubated with 32P-la- beled pIRE probe to assess IFN-inducible ISGF3-independent STAT2:1 hetero- dimer formation and DNA binding. C, chromatin binding activity of complexes containing either intact STAT2 or the VV-II STAT2. Cells were untreated () or treated () with 5 ng/ml IFN alfacon-1 for 30 min. Following cell lysis and DNA son- ication, aliquots were collected and used as input samples. ChIPs were then per- formed when anti-STAT2 antibody was either added or omitted, as indicated. The precipitated chromatin was analyzed us- ing primers specific for a 6–16 ISRE and an IRF-1 GAS. Primers for -actin were used to confirm the immunoprecipitation is specific for STAT2. D, the signal inten- sity of each band was determined. Histo- grams representing signal intensity ra- tios of each ChIP sample band to its corresponding input band for the 6–16 and IRF-1 primer sets are provided. Data are representative of three independent experiments.

Article Snippet: Antibodies against phospho-STAT2 and phospho-STAT1 were obtained from Cell Signaling Technology (Beverly, MA).

Techniques: Binding Assay, Incubation, Labeling, Positive Control, Nucleic Acid Electrophoresis, Autoradiography, Activity Assay, Lysis, Immunoprecipitation

Journal: The Journal of Experimental Medicine

Article Title: Hes1 attenuates type I IFN responses via VEGF-C and WDFY1

doi: 10.1084/jem.20180861

Figure Lengend Snippet: Hes1 deficiency results in enhanced TLR3-induced expression of type I IFN and ISGs. (A) Heatmap of superinduced genes by poly(I:C) in Hes1 fl/fl Cre-ER T2 (Hes1 KO) BMDMs versus Hes1 +/+ Cre-ER T2 (WT) cells. BMDMs were stimulated without or with 1 µg/ml poly(I:C) for the indicated periods. Ifnb1 in listed genes is highlighted in red. FC, fold change. (B) Percentage of ISGs (defined on the Interferome database) among all superinduced genes by poly(I:C) at 3 h in Hes1 KO BMDMs. (C) qPCR analysis of ISG mRNA in WT and Hes1 KO BMDMs stimulated with poly(I:C) for 3 h. (D) qPCR analysis of Mx1 in WT and Hes1 KO BMDMs stimulated with IFN-β (10 U/ml) for 3 h. (E) Immunoblotting analysis of phosphorylated (p-) and total STAT1 (Tyr701) and STAT2 (Tyr689) in whole-cell lysates of WT and Hes1 KO BMDMs stimulated with poly(I:C) for various times (top lanes). Levels of p38α served as loading controls. (F) qPCR analysis of Ifnb1 in WT and Hes1 KO BMDMs stimulated with poly(I:C) for various times (left). Cumulative results of Ifnb1 expression are shown (right). (G) qPCR analysis of Ifnb1 and Mx1 in WT and Hes1 KO BMDMs stimulated with various concentrations of poly(I:C) (horizontal axes) for 2 h. (H) ELISA of IFN-β in supernatant of WT and Hes1 KO BMDMs stimulated with poly(I:C) for various times (horizontal axes). Data are representative of one (A and B) or three independent experiments (C–E, F [left], and G; mean + SD of technical triplicates in C, D, F [left], and G) or are pooled from three (F [right] and H; mean ± SD in H) independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Student’s t test).

Article Snippet: Antibodies against STAT2 (1:1,000, 72604), p-STAT1 (1:1,000, 7649), p-ERK (1:1,000, 9101), ERK (1:1,000, 9102), p-p38 (1:1,000, 9215), p-p65 (1:1,000, 3033), p65(1:1,000, 4764), IκBα (1:1,000, 4812), TBK1 (1:1,000, 3013), p-JNK (1:1,000, 9251), p-TBK1 (1:1,000, 5483), p-IKKε (1:1,000, 8766), IKKε (1:1,000, 3416), and p-IRF3 (1:500, 4947) were purchased from Cell Signaling Technology.

Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: The Brazilian Journal of Infectious Diseases

Article Title: Antiviral and myocyte protective effects of IL-28A in coxsackievirus B3-induced myocarditis

doi: 10.1016/j.bjid.2014.10.007

Figure Lengend Snippet: IL-28A increased the expression of STAT1 and STAT2 in heart tissue. Uninfected and CVB3-infected mice were treated with or without IL-28A (40 μg/kg) for 4 days, then the heart tissues of infected mice were removed on day 4 post-infection and subjected to Western blot analysis. Actin was used as a loading control. The amount of STAT2, STAT1 and Actin was quantified using NIH Image J and the ratio of STAT2 or STAT1 to Actin was graphed in the lower panels. IL-28A treatment resulted in a significant increase in the expression of STAT1 and STAT2 compared to infected group (** p < 0.01).

Article Snippet: The membranes were blocked in 5% BSA for 40 min at room temperature and then incubated individually at 4 °C overnight with primary antibodies, including rabbit polyclonal antibodies against STAT1 (1:1000) and actin (1:2000), and mouse monoclonal antibodies against STAT2 (1:200) (Cell Signaling Technology, Inc., Danvers, MA).

Techniques: Expressing, Infection, Western Blot, Control

Journal: Scientific Reports

Article Title: Integrated Genomics Identifies Convergence of Ankylosing Spondylitis with Global Immune Mediated Disease Pathways

doi: 10.1038/srep10314

Figure Lengend Snippet: ( A ) Interaction network of STAT2 and STAT3 gene with other non-HLA risk genes identified in nine immune mediated diseases. ( B ) Gene expression levels of STAT3 and STAT2 in AS cases and controls. A qPCR analysis of patient-derived B-Cell lines showed the relative expression of STAT2 and STAT3 genes across the affected (white bar) and unaffected individuals (black bar). Individuals are also grouped according to their clinical diagnosis to compare the expression, normalized to GAPDH only. The bar plot represents the mRNA expression level (Y-axis) and upper and lower limit represented by mean ± SEM. A t-test confirmed significant differences ( p < 0.05) between the groups for both genes.

Article Snippet: For gel running samples were diluted with water and loading buffer to a final concentration of 1-2 ug Protein/ul and denatured by boiling for 6 min. 20 ug protein samples were loaded per lane on a precast TGX polyacrylamide gels (4-20%, BioRad), along with a lane containing Precision Plus Dual Color Protein Standards (BioRad) and electrophoresed for 1.5 hr at 100 V. Transfer to nitrocellulose was performed for 1hr at 100 V. Blots were blocked with 5%milk/TBS-T for 1 hr, probed with antibodies against STAT2 (Genscript A01263), STAT3 (Cel Sign.

Techniques: Gene Expression, Derivative Assay, Expressing, Biomarker Discovery