total stat2  (Cell Signaling Technology Inc)

Journal: Viruses

Article Title: Human Platelet Lysate Induces Antiviral Responses against Parechovirus A3

doi: 10.3390/v14071499

Figure Lengend Snippet: hPL-induced innate immunity response type I interferon and antiviral-related genes on GBM cells. ( A ) GBM cells were cultured under FBS, 2.5% and 5% hPL for 6 h, 24 h, and 48 h. The cell protein lysates were employed to analyze the innate immunity pathways by immunoblotting using anti-phospho-STAT1, anti-total STAT2, anti-phospho-STAT2, anti-total STAT2, anti-phospho-IRF3, anti-total IRF3, anti-phospho-NFκB, anti-total NFκB p65, and anti-GAPDH. ( B ) Quantification of protein expressions was normalized to GAPDH. Data are mean ± SD of three independent experiments. Student’s t -test, *, p < 0.05; **, p <0.01 compared with GBM cells supplemented with FBS. ( C ) GBM cells were cultured under FBS, 2.5% and 5% hPL mediums for 24 h and 48 h. The mRNA level of antiviral-related genes IFN-α , IFN-β , IRF3 , and MxA were confirmed by qPCR. The level of transcripts was normalized to GAPDH. Data are mean ± SD of three independent experimental samples. Student’s t -test, *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with FBS.

Article Snippet: The antibodies were anti-PeV VP0 (LTK BioLaboratories, Taoyuan, Taiwan) [ , ], anti-phospho STAT2 (Cell Signaling, 88410, Danvers, MA, USA), anti-total STAT2 (Cell Signaling, #72604), anti-phospho STAT1 (Cell Signaling, #9167), anti-total STAT1 (Cell Signaling, #14994), anti-phospho IRF3 (Abcam, #ab76493, Cambridge, England), anti-total IRF3 (Cell Signaling, #11904), anti-phospho NFκB p65 (Abcam, #ab86299), anti-total NFκB p65 (Cell Signaling, #8242) and anti-GAPDH (Proteintech, 60004-1-Ig, Rosemont, IL, USA).

Techniques: Cell Culture, Western Blot

Journal: Viruses

Article Title: Human Platelet Lysate Induces Antiviral Responses against Parechovirus A3

doi: 10.3390/v14071499

Figure Lengend Snippet: hPL-supplemented growth medium-induced expression of IFN-signaling pathway under PeV-A3 infection of GBM cells. ( A ) GBM cells were infected with PeV-A3 at MOI = 1 for 6 h, 24 h, and 48 h under FBS- or hPL-supplemented growth mediums. Cellular protein lysates were extracted to analyze IFN-signaling pathway by immunoblotting using anti-PeV VP0, anti-phospho-STAT1, anti-total STAT1, anti-phospho-STAT2, anti-total STAT2, anti-phospho-IRF3, anti-total IRF3, anti-phospho-NFκB, anti-total NFκB p65, and anti-GAPDH. ( B ) Quantification of different protein expressions was normalized to GAPDH. Data are mean ± SD of three independent samples. Student’s t -test, *, p < 0.05 compared with FBS.

Article Snippet: The antibodies were anti-PeV VP0 (LTK BioLaboratories, Taoyuan, Taiwan) [ , ], anti-phospho STAT2 (Cell Signaling, 88410, Danvers, MA, USA), anti-total STAT2 (Cell Signaling, #72604), anti-phospho STAT1 (Cell Signaling, #9167), anti-total STAT1 (Cell Signaling, #14994), anti-phospho IRF3 (Abcam, #ab76493, Cambridge, England), anti-total IRF3 (Cell Signaling, #11904), anti-phospho NFκB p65 (Abcam, #ab86299), anti-total NFκB p65 (Cell Signaling, #8242) and anti-GAPDH (Proteintech, 60004-1-Ig, Rosemont, IL, USA).

Techniques: Expressing, Infection, Western Blot

Journal: Journal of Molecular Endocrinology

Article Title: Temporal regulation of interferon signalling in human EndoC-βH1 cells

doi: 10.1530/JME-21-0224

Figure Lengend Snippet: EndoC-βH1 cells were treated with various interferons (IFNα – 1000 U/mL; IFNɤ – 20 ng/mL; IFNʎ1 or 2 – 200 ng/mL) for either 30 min or 24 h. Protein was extracted at the end of the incubation and Western blotting performed using anti-phospho STAT1, anti STAT1, anti-phospho STAT2 and anti STAT2. A representative blot is shown for each target protein, but densitometric traces were obtained from a minimum of three separate experiments in each case (lower panels). GAPDH was used as a loading control. **** P < 0.0001; ** P < 0.01; * P < 0.05.

Article Snippet: Fixed cells were permeabilised with buffers containing 0.2% Triton (0.1 M lysine, 10% donor calf serum, 0.02% sodium azide, in PBS) for 30 min prior to staining with either anti-total STAT1 (Cell Signalling #14994; 1 in 200 dilution) and/or anti-total STAT2 antibody (Thermofisher Scientific #44-362G; 1 in 200 dilution).

Techniques: Incubation, Western Blot, Control

Journal: Journal of Molecular Endocrinology

Article Title: Temporal regulation of interferon signalling in human EndoC-βH1 cells

doi: 10.1530/JME-21-0224

Figure Lengend Snippet: EndoC-βH1 were treated with IFNα (1000 U/mL) or IFNɤ for 24 h, as shown. The cells were washed with PBS and the medium replaced with normal medium (lacking interferons) and cultured for increasing periods of time (24, 48, 96 and 144 h). Protein was extracted at each time point and Western blotting performed using anti-STAT1 and anti-STAT2 sera as shown. Representative blots are presented and GAPDH was used as a loading control.

Article Snippet: Fixed cells were permeabilised with buffers containing 0.2% Triton (0.1 M lysine, 10% donor calf serum, 0.02% sodium azide, in PBS) for 30 min prior to staining with either anti-total STAT1 (Cell Signalling #14994; 1 in 200 dilution) and/or anti-total STAT2 antibody (Thermofisher Scientific #44-362G; 1 in 200 dilution).

Techniques: Cell Culture, Western Blot, Control

Journal: Journal of Molecular Endocrinology

Article Title: Temporal regulation of interferon signalling in human EndoC-βH1 cells

doi: 10.1530/JME-21-0224

Figure Lengend Snippet: EndoC-βH1 were treated with IFNα (1000 U/mL) or IFNγ (20 ng/mL) or IFNʎ1 or 2 (200 ng/mL) for 24 h. After this period, cells were lysed and 2% of the input lysate removed, denatured and stored at −20°C. The remainder of the protein lysate was incubated with 2 µg of STAT1 antibody or 2 µg of isotype control IgG overnight at 4°C. A 50% bead slurry was added, proteins eluted and Western blotting was performed using antisera directed against either STAT2 and STAT1.

Article Snippet: Fixed cells were permeabilised with buffers containing 0.2% Triton (0.1 M lysine, 10% donor calf serum, 0.02% sodium azide, in PBS) for 30 min prior to staining with either anti-total STAT1 (Cell Signalling #14994; 1 in 200 dilution) and/or anti-total STAT2 antibody (Thermofisher Scientific #44-362G; 1 in 200 dilution).

Techniques: Incubation, Control, Western Blot

Journal: Journal of Molecular Endocrinology

Article Title: Temporal regulation of interferon signalling in human EndoC-βH1 cells

doi: 10.1530/JME-21-0224

Figure Lengend Snippet: EndoC-βH1 treated with either 1000 U/mL IFNα, 20 ng/mL IFNγ or 200 ng/mL IFNʎ1 or IFNʎ2 for 30 min. Cells were fixed with 4% paraformaldehyde, permeabilised and stained with anti-STAT1 or anti-STAT2 antisera, as shown. Images were taken using a Leica DM4000 B LED Fluorescence microscope using exposure settings defined for the control conditions (upper and middle panels of each figure). In the lower panels of each figure, the exposure settings were adjusted to provide improved resolution of the subcellular localisation of each STAT isoform after incubation with interferons.

Article Snippet: Fixed cells were permeabilised with buffers containing 0.2% Triton (0.1 M lysine, 10% donor calf serum, 0.02% sodium azide, in PBS) for 30 min prior to staining with either anti-total STAT1 (Cell Signalling #14994; 1 in 200 dilution) and/or anti-total STAT2 antibody (Thermofisher Scientific #44-362G; 1 in 200 dilution).

Techniques: Staining, Fluorescence, Microscopy, Control, Incubation

Journal: Journal of Molecular Endocrinology

Article Title: Temporal regulation of interferon signalling in human EndoC-βH1 cells

doi: 10.1530/JME-21-0224

Figure Lengend Snippet: EndoC-βH1 were treated with either IFNα – 1000 U/mL or IFNγ – 20 ng/mL for 24 h. Cells were washed with PBS and retreated with either IFNα (1000 U/mL) or IFNγ (20 ng/mL) for a further of 30 min. Protein was extracted at the end of each incubation period and Western blotting performed using anti-pSTAT1, anti-total STAT1, anti-pSTAT2 and anti-total STAT2. Representative blots are presented but densitometric traces were obtained from a minimum of three separate experiments in each case (lower panels). GAPDH was used as a loading control. **** P < 0.0001; ** P < 0.01; * P < 0.05.

Article Snippet: Fixed cells were permeabilised with buffers containing 0.2% Triton (0.1 M lysine, 10% donor calf serum, 0.02% sodium azide, in PBS) for 30 min prior to staining with either anti-total STAT1 (Cell Signalling #14994; 1 in 200 dilution) and/or anti-total STAT2 antibody (Thermofisher Scientific #44-362G; 1 in 200 dilution).

Techniques: Incubation, Western Blot, Control

Journal: Journal of Molecular Endocrinology

Article Title: Temporal regulation of interferon signalling in human EndoC-βH1 cells

doi: 10.1530/JME-21-0224

Figure Lengend Snippet: EndoC-βH1 were treated with either IFNα (1000 U/mL) or IFNλ1 or IFNλ2 (20 ng/mL) for 24 h. After this time, cells were washed and retreated with either IFNα (1000 U/mL) or IFNλ1 or IFNλ2 (20 ng/mL) for a further 30 min. Protein was extracted at the end of the incubation and Western blotting performed using anti-pSTAT1, anti-total STAT1, anti-pSTAT2 and anti-total STAT2. GAPDH was used as a loading control (representative blots are shown; n = 3).

Article Snippet: Fixed cells were permeabilised with buffers containing 0.2% Triton (0.1 M lysine, 10% donor calf serum, 0.02% sodium azide, in PBS) for 30 min prior to staining with either anti-total STAT1 (Cell Signalling #14994; 1 in 200 dilution) and/or anti-total STAT2 antibody (Thermofisher Scientific #44-362G; 1 in 200 dilution).

Techniques: Incubation, Western Blot, Control

Journal: Journal of Molecular Endocrinology

Article Title: Temporal regulation of interferon signalling in human EndoC-βH1 cells

doi: 10.1530/JME-21-0224

Figure Lengend Snippet: EndoC-βH1 were treated with either IFNγ (1000 U/mL) or IFNλ1 or IFNλ2 (20 ng/mL) for 24 h. After this time, cells were washed and retreated with either IFNγ (1000 U/mL) or IFNλ1 or IFNλ2 (20 ng/mL) for a further 30 min. Protein was extracted at the end of the incubation and Western blotting performed using anti-pSTAT1, anti-total STAT1, anti-pSTAT2 and anti-total STAT2. GAPDH was used as a loading control (representative blots are shown; n = 3).

Article Snippet: Fixed cells were permeabilised with buffers containing 0.2% Triton (0.1 M lysine, 10% donor calf serum, 0.02% sodium azide, in PBS) for 30 min prior to staining with either anti-total STAT1 (Cell Signalling #14994; 1 in 200 dilution) and/or anti-total STAT2 antibody (Thermofisher Scientific #44-362G; 1 in 200 dilution).

Techniques: Incubation, Western Blot, Control

Journal: Journal of Molecular Endocrinology

Article Title: Temporal regulation of interferon signalling in human EndoC-βH1 cells

doi: 10.1530/JME-21-0224

Figure Lengend Snippet: EndoC-βH1 were treated with increasing concentration of IFNα (1, 10, 100 or 1000 U/mL) for 24 h. Cells were then washed with PBS and treated with 1000 U/mL IFNα for a further of 30 min. Cells were lysed and Western blotting performed using anti-pSTAT1, anti-total STAT1, anti-pSTAT2 and anti-total STAT2. GAPDH was used as a loading control. Representative blots are presented but densitometric traces were obtained from two separate experiments in each case (lower panels). **** P < 0.0001; *** P < 0.001; ** P < 0.01; * P < 0.05.

Article Snippet: Fixed cells were permeabilised with buffers containing 0.2% Triton (0.1 M lysine, 10% donor calf serum, 0.02% sodium azide, in PBS) for 30 min prior to staining with either anti-total STAT1 (Cell Signalling #14994; 1 in 200 dilution) and/or anti-total STAT2 antibody (Thermofisher Scientific #44-362G; 1 in 200 dilution).

Techniques: Concentration Assay, Western Blot, Control