Journal: Molecular Metabolism

Article Title: Baricitinib counteracts metaflammation, thus protecting against diet-induced metabolic abnormalities in mice

doi: 10.1016/j.molmet.2020.101009

Figure Lengend Snippet: Baricitinib attenuates the HD-induced JAK-STAT pathway. Western blotting analysis for phosphorylation of Tyr 1007/1008 JAK1/2 in the skeletal muscle (A) and kidneys (B) and normalized to total JAK1/2 and for phosphorylation of Tyr 690 on STAT2 in the skeletal muscle (C) and kidneys (D) and normalized to total STAT2. All of the data are expressed as mean ± SEM for n = 6 per group. ∗p< 0.05 vs ND and •p< 0.05 vs HD.

Article Snippet: The antibodies used were rabbit anti-Tyr 1007/1008 JAK2 (#3776), rabbit anti-total JAK2 (#3230), rabbit anti-Tyr 690 STAT2 (Bioss Antibodies, bs-3428R), rabbit anti-total STAT2 (#72604), rabbit p21 (#2947), rabbit anti-Ser 307 IRS-1 (#2381), mouse anti-total IRS-1 (#3194), rabbit anti-Ser 473 AKT (#4060), rabbit anti-total AKT (#9272), rabbit anti-Ser 9 GSK-3β (#9332) and rabbit anti-total GSK-3β (9315).

Techniques: Western Blot

Journal:

Article Title: Sendai Virus Blocks Alpha Interferon Signaling to Signal Transducers and Activators of Transcription

doi:

Figure Lengend Snippet: Poor induction of ISG products in SeV-infected cells. HeLa cells were mock infected (lanes 1 to 3) or infected with SeV (lanes 4 to 9), and then the media were replaced at 2 hpi with fresh medium containing 103 IU of IFN-α per ml (lanes 2, 3, 5, and 6) or no IFN-α (lanes 1, 4, 7, 8, and 9). The cells were harvested at 0 (lanes 1, 4, and 7), 8 (lanes 2, 5, and 8), and 24 (lanes 3, 6, and 9) h after IFN-α treatment. Total-cell extracts (20 μg of protein) prepared according to the method of Lee et al. (14) were subjected to Western blot analysis as described previously (9). Anti-PKR rabbit polyclonal (sc-707) (A), anti-Stat1 mouse monoclonal (sc-464) (B), anti-Stat2 rabbit polyclonal (sc-476) (C), and anti-p48 rabbit polyclonal (sc-496) (D) antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.) were used as the first antibody. The same blotting membrane was stripped and reprobed. The protein concentration was determined as described previously (9).

Article Snippet: Anti-PKR rabbit polyclonal (sc-707) (A), anti-Stat1 mouse monoclonal (sc-464) (B), anti-Stat2 rabbit polyclonal (sc-476) (C), and anti-p48 rabbit polyclonal (sc-496) (D) antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.) were used as the first antibody.

Techniques: Infection, Western Blot, Protein Concentration

Journal:

Article Title: Sendai Virus Blocks Alpha Interferon Signaling to Signal Transducers and Activators of Transcription

doi:

Figure Lengend Snippet: Inhibition of IFN-α-stimulated tyrosine phosphorylation of Stat1, Stat2, and Stat3 in SeV-infected cells. HeLa cells were mock infected (lanes 1 to 3) or infected (lanes 4 to 9) with SeV at 2 h prior to replacement with fresh medium containing 103 IU of IFN-α per ml (lanes 2, 3, 5, and 6) or no IFN-α (lanes 1, 4, 7, 8, and 9). The cells were harvested at 0 (lanes 1, 4, and 7), 5 (lanes 2, 5, and 8), or 30 (lanes 3, 6, and 9) min after IFN-α treatment. Total-cell extracts (50 μg of protein) were subjected to Western blot analysis with anti-phospho-(Tyr 701)-Stat1 (no. 9171) (A) and anti-phospho-(Tyr 705)-Stat3 (no. 9131) (C) rabbit polyclonal antibodies (New England Biolabs, Inc.). To detect tyrosine-phosphorylated Stat2, total-cell extracts (500 μg) were immunoprecipitated with an anti-Stat2 antibody (sc-476) (B) before Western blot analysis with antiphosphotyrosine mouse monoclonal antibody (sc-7020) (Santa Cruz Biotechnology, Inc.). Each blotting membrane was stripped and reprobed with anti-Stat1 (sc-464) (A) mouse monoclonal antibody or anti-Stat2 (sc-476) (B) or anti-Stat3 (sc-7179) (C) rabbit polyclonal antibody (Santa Cruz Biotechnology, Inc.).

Article Snippet: Anti-PKR rabbit polyclonal (sc-707) (A), anti-Stat1 mouse monoclonal (sc-464) (B), anti-Stat2 rabbit polyclonal (sc-476) (C), and anti-p48 rabbit polyclonal (sc-496) (D) antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.) were used as the first antibody.

Techniques: Inhibition, Infection, Western Blot, Immunoprecipitation

Journal: Journal of Virology

Article Title: Equid Herpesvirus 1 Targets the Sensitization and Induction Steps To Inhibit the Type I Interferon Response in Equine Endothelial Cells

doi: 10.1128/JVI.01342-19

Figure Lengend Snippet: Effect of EHV-1 infection on IFN-induced STAT1/STAT2 phosphorylation. EECs were either mock infected (M) or infected with T953 (E1) at an MOI of 5, and cells were either treated with equine rEqIFN-α at 1,000 IU/ml or left untreated (+ or −, respectively) for 30 min before lysis. Abundance of phosphorylated versus total STAT1 at 12 h (A) and phosphorylated versus total STAT2 at 3 h (B), 6 h (C), or 12 h (D) were then quantified using Western blot analysis. Membranes were stripped and reprobed with β-actin as a control for equal loading of samples. Densitometric analysis of Western blot images for phosphorylated STAT1 and total STAT1 (E) and phosphorylated STAT2 and total STAT2 (F) normalized against β-actin was conducted using ImageJ (NIH). The Western blot images are representative of 3 different blots from independent experiments.

Article Snippet: Rabbit anti-phospho-STAT2 primary antibody was purchased from Rockland Immunochemicals Inc. (Limerick, PA).

Techniques: Infection, Lysis, Western Blot

Journal: Journal of Virology

Article Title: Equid Herpesvirus 1 Targets the Sensitization and Induction Steps To Inhibit the Type I Interferon Response in Equine Endothelial Cells

doi: 10.1128/JVI.01342-19

Figure Lengend Snippet: Effect of EHV-1 infection on nuclear accumulation of STAT2. EECs grown on coverslips in 24-well plates were either infected with T953 at an MOI of 3 or mock infected. The cells were either stimulated with equine rEqIFN-α at 1,000 IU/ml or treated with an equal volume of plain medium for 30 min prior to fixation in 4% PFA at 12 hpi. Cells were stained with anti-STAT2 (1:100 dilution) and anti-EHV-1 gC antibody (1:200 dilution). Approximately 300 cells from different fields were examined for each treatment using an inverted fluorescence microscope. (A) Mock-infected EECs treated with plain medium and stained for STAT2. (B) Mock-infected cells stimulated with equine rEqIFN-α and stained for STAT2. (C) T953-infected cells stained for STAT2. (D) T953-infected cells stimulated with equine rEqIFN-α and stained for STAT2. 4,6-Diamidino-2-phenylindole (DAPI; blue) served as a counterstain. Solid arrows indicate representative cells without nuclear STAT2 translocation, whereas open arrows indicate representative cells with nuclear STAT2 translocation. Scale bars, 50 μm. (E) Quantification of nuclear STAT2 intensity of IF images using Nikon NIS-Elements software as described in Materials and Methods. (F) EECs were either mock infected (M) or infected with T953 (E1) at an MOI of 3, and cells were either treated with equine rEqIFN-α (1,000 IU/ml) or left untreated (+ or −, respectively) for 30 min before fractionation into nuclear (N) or cytoplasmic (C) compartments. Separated cellular fractions were probed for phosphorylated STAT2 and total STAT2 to determine their cellular distribution following T953 infection. Lamin A/C and MEK1/2 were used as nuclear and cytoplasmic loading controls, respectively.

Article Snippet: Rabbit anti-phospho-STAT2 primary antibody was purchased from Rockland Immunochemicals Inc. (Limerick, PA).

Techniques: Infection, Staining, Fluorescence, Microscopy, Translocation Assay, Software, Fractionation

Journal: Journal of Virology

Article Title: Equid Herpesvirus 1 Targets the Sensitization and Induction Steps To Inhibit the Type I Interferon Response in Equine Endothelial Cells

doi: 10.1128/JVI.01342-19

Figure Lengend Snippet: Effect of UV inactivation on type I IFN molecules. Mock-infected EECs (M, solid bars) were treated with either 80 μg/ml of poly(I·C) (A, C, and D) or 10 μg/ml of LPS (B) (both labeled as P, white bars) or infected with UV-inactivated T953 in the absence (E1, checkerboard bars) or presence (P+E1, diagonally striped bars) of P as described in the text. At the indicated time points, the cells were lysed and equine TLR3 (A), TLR4 (B), IRF7 (C), and IRF9 mRNA (D) were quantified by real-time RT-PCR. Data were normalized to the levels of endogenous control equine RPLP0 mRNA at the same time point. ns, not significant. (E) EECs were either mock infected (M) or infected with UV-inactivated T953 (E1) at an MOI of 5, and cells were either treated with 1,000 IU/ml of equine rEqIFN-α or left untreated (+ or −, respectively) for 30 min prior to lysis. At 12 hpi, cells were lysed, and equal amounts of proteins were separated on 10% SDS-PAGE and probed using phospho-STAT2 or STAT2. Images are representative of 3 independent biological experiments.

Article Snippet: Rabbit anti-phospho-STAT2 primary antibody was purchased from Rockland Immunochemicals Inc. (Limerick, PA).

Techniques: Infection, Labeling, Quantitative RT-PCR, Lysis, SDS Page

Journal: Journal of Virology

Article Title: Equid Herpesvirus 1 Targets the Sensitization and Induction Steps To Inhibit the Type I Interferon Response in Equine Endothelial Cells

doi: 10.1128/JVI.01342-19

Figure Lengend Snippet: Effect of viral late gene blockage on STAT2 activation. EECs were infected with T953 (E1) either in the presence (+) or absence (−) of PAA (300 μg/ml). Mock-infected EECs (M) were either treated with equine rEqIFN-α at 1,000 IU/ml or left untreated (+ or −, respectively) for 30 min before lysis. Abundance of phosphorylated versus total STAT2 at 3 h (A), 6 h (B), or 12 h (C) was then quantified using Western blot analysis. Membranes were stripped and reprobed with β-actin as a control for equal loading of samples. As controls for effective PAA-mediated blockage of T953 L proteins, both the EHV-1 IE and EHV-1 gD were also quantified. The Western blot images are representative of 3 different blots from independent experiments.

Article Snippet: Rabbit anti-phospho-STAT2 primary antibody was purchased from Rockland Immunochemicals Inc. (Limerick, PA).

Techniques: Activation Assay, Infection, Lysis, Western Blot

Journal: Journal of Virology

Article Title: Equid Herpesvirus 1 Targets the Sensitization and Induction Steps To Inhibit the Type I Interferon Response in Equine Endothelial Cells

doi: 10.1128/JVI.01342-19

Figure Lengend Snippet: Schematic illustration of our proposal of how EHV-1 T953 strain blocks the key molecules required for type I IFN production. (A) During the sensitization phase of host type I IFN production, EHV-1 blocks the expression of TLR3 and TLR4 mRNA, thereby enabling the virus to avoid host detection. (B) In the subsequent induction phase, EHV-1 specifically degrades the cellular level of TYK2 protein. This consequently blocks the abundance of cellular levels of phosphorylated/activated STAT1 and STAT2 molecules in infected cells. At the same time, EHV-1 blocks the transcription of IRF9 mRNA and thus prevents the formation of ISGF3 complex needed to transactivate ISRE for ISG production. EHV-1 also inhibits the transcription of IRF7 mRNA, exerting a negative effect on IFN-α production. The overall effect of EHV-1 renders the host cell unable to restrict viral spread by direct cell-to-cell contact during infection.

Article Snippet: Rabbit anti-phospho-STAT2 primary antibody was purchased from Rockland Immunochemicals Inc. (Limerick, PA).

Techniques: Expressing, Infection