anti p stat3 primary antibody solution  (Cell Signaling Technology Inc)

Journal: Protein & Cell

Article Title: IL-24 promotes atopic dermatitis-like inflammation through driving MRSA-induced allergic responses

doi: 10.1093/procel/pwae030

Figure Lengend Snippet: IL-24 activates JAK-STAT pathway in keratinocytes. (A) Characterization of different signaling pathways determined by luciferase reporter assay in HEK293T cells co-expressing the receptors. AP-1, activator protein-1; CREB, cyclic-AMP response binding protein; IFN-β, interferon-β; ISRE, interferon-stimulated response elements; m67-SIE, m67-sis inducible element; NF-κB, nuclear factor-κB. n = 4, mean ± SEM. *** P < 0.001 by unpaired t -test. ns, not significant. (B) Luciferase reporter assay of m67-SIE responding to rhIL-24 or rmIL-24 stimulation in HEK293T cells co-expressing the receptors. Relative luciferase activities driven by m67-SIE were measured at 12 h post rIL-24 treatment. n = 4, mean ± SEM. **** P < 0.0001 by unpaired t -test. ns, not significant. (C) Immunoblot analysis of HEK293T cells expressing indicated receptors responding to rhIL-24 stimulation. (D) Immunoblot analysis of skin samples from wild-type mice treated with indicated rIL-24 or vehicle-control. (E–H) Representative immunofluorescent images showing the skin of rhIL-24-treated group and vehicle-treated control group for 1 h. Zoomed views on the right indicate the regions outlined by orange dashed rectangles. Quantification was shown in (F and H) indicating the phosphorylation of STAT3 and STAT1, respectively. Antikeratin 14 antibody was used to label K14 + keratinocytes. n = 3–4 mice, mean ± SEM. * P < 0.05, *** P < 0.001 by unpaired t -test. Dashed lines indicate the border of epidermis.

Article Snippet: Primary antibodies include rabbit anti-p-STAT1 (Cell Signaling Technology; Cat# 9167; RRID: AB_561284 ), rabbit anti-p-STAT3 (Cell Signaling Technology; Cat# 9145; RRID: AB_2491009 ), rabbit anti-STAT3 (Cell Signaling Technology; Cat# 12640; RRID: AB_2629499 ), rabbit anti-GAPDH (Cell Signaling Technology; Cat# 5174; RRID: AB_10622025 ), rabbit anti-β-Actin (BioLegend; Cat# 622102; RRID: AB_315946 ), mouse antikeratin 14 (Santa Cruz Biotechnology; Cat# sc-53253; RRID: AB_2134820 ).

Techniques: Protein-Protein interactions, Luciferase, Reporter Assay, Expressing, Binding Assay, Western Blot, Control, Phospho-proteomics

Journal: Frontiers in Immunology

Article Title: HSP90AA1 interacts with CSFV NS5A protein and regulates CSFV replication via the JAK/STAT and NF-κB signaling pathway

doi: 10.3389/fimmu.2022.1031868

Figure Lengend Snippet: HSP90AA1 over-expression activates STAT1 and P65 in PK-15 and 3D4/2 cells. (A) HSP90AA1 over-expression affected the phosphorylation of STAT1 and nuclear translocation of p-STAT1 in PK-15 and 3D4/2 cells were observed by laser confocal microscopy. (B) HSP90AA1 over-expression affected the nuclear translocation of p-P65 in PK-15 and 3D4/2 cells were observed by laser confocal microscopy.

Article Snippet: Primary antibodies include rabbit anti-p-STAT1 pAb (Bioss, bs-3427R), rabbit-anti-p-P65 pAb (Affinity Biosciences, AF2006) and mouse anti-Flag mAb.

Techniques: Over Expression, Translocation Assay, Confocal Microscopy

Journal: Frontiers in Immunology

Article Title: HSP90AA1 interacts with CSFV NS5A protein and regulates CSFV replication via the JAK/STAT and NF-κB signaling pathway

doi: 10.3389/fimmu.2022.1031868

Figure Lengend Snippet: CSFV infection antagonizes the activation of HSP90AA1 on JAK/STAT pathway. (A, B) Western blot for JAK1, STAT1, p-JAK1, p-STAT1, OAS2, ISG-15 and HSP90AA1 expression in HSP90AA1-overexpression or knock-downed PK-15 and 3D4/2 cells. Cells were infected with CSFV (MOI=1) after transfection p3×Flag-HSP90AA1 or siHSP90AA1. The cells were not infected with CSFV as a control. Cells were harvested at 24hpi and 48hpi served to Western blot. The relative levels of proteins were estimated by histograms representing density reading of the gel bands with Image J, and the ratios were calculated relative to tubulin control. (*p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 calculated using two-way ANOVA, ns, not significant).

Article Snippet: Primary antibodies include rabbit anti-p-STAT1 pAb (Bioss, bs-3427R), rabbit-anti-p-P65 pAb (Affinity Biosciences, AF2006) and mouse anti-Flag mAb.

Techniques: Infection, Activation Assay, Western Blot, Expressing, Over Expression, Transfection

Journal: Viruses

Article Title: INMI1 Zika Virus NS4B Antagonizes the Interferon Signaling by Suppressing STAT1 Phosphorylation

doi: 10.3390/v13122448

Figure Lengend Snippet: ZIKV INMI1 NS4B inhibits STAT1 phosphorylation. ( a ) Inhibition of pISRE-luc expression in HEK293T cells transfected with increasing concentrations of ZIKV NS2A (red line), NS4B (green line) or the combination of the two plasmids (blue line), and stimulated with IFN-α (10 ng/mL). Results are shown as percentages of pISRE-luc activation in NS2A or NS4B transfected cells over empty vector (EV) transfected controls; ( b ) the isobologram visualizes the synergistic effect of the combination of ZIKV NS2A and NS4B at their respective EC 50 ; ( c ) immunofluorescence of HEK293T transfected for 24 h and 36 h with ZIKV NS2A (row 1 and 3) and NS4B (row 2 and 4) and stimulated with IFN-α; FLAG (green) and P-STAT1 (red) signals are detected; nuclei (blue) are stained with Hoechst; scale bar, 10 μm; ( d ) quantification was performed counting positive cells for nuclear P-STAT1 (red signal) in EV, NS2A and NS4B transfected cells, 6–10 fields per each condition, from a total of 3 biologically independent experiments, were analyzed; cells count was performed using Cell Counter plugin of the image analysis program ImageJ, **** p < 0.0001 as obtained by unpaired t test. ( e ) Western blot of HEK293T transfected with empty vector (EV) (lane 1–2) or ZIKV NS4B (lane 3–4) stimulated (+) (lanes 2–4) or not (−) (lanes 1–3) with IFN-α; membranes were stained for P-STAT1, STAT1-tot and GAPDH antibodies; ( f ) Western blot of HEK293T transfected with empty vector (EV) (lane 1–2) or ZIKV NS4B (lane 3–4); cells were treated (+) (lanes 2–4) or not (−) (lanes 1–3) with MG132; blot membranes were stained for STAT1-tot and GAPDH antibodies.

Article Snippet: Membranes were probed with monoclonal rabbit primary antibodies against P-STAT1 (1:1000) and GAPDH (1:1000) (Cell Signaling, Denver, MA, USA), and polyclonal rabbit primary antibody against STAT1-tot (1:1000) (Cell Signaling, Denver, MA, USA).

Techniques: Phospho-proteomics, Inhibition, Expressing, Transfection, Activation Assay, Plasmid Preparation, Immunofluorescence, Staining, Western Blot

Journal: Viruses

Article Title: INMI1 Zika Virus NS4B Antagonizes the Interferon Signaling by Suppressing STAT1 Phosphorylation

doi: 10.3390/v13122448

Figure Lengend Snippet: ZIKV INMI1 NS4B blocks STAT2 nuclear transport. ( a ) Immunofluorescence of HEK293T cells transfected with empty vector (EV) or ZIKV NS2A (row 3) stimulated with IFN-α. FLAG (green) and P-STAT2 (red) signals were detected; nuclei (blue) were stained with Hoechst; scale bar, 10 μm; ( b ) quantification was performed counting positive cells for nuclear P-STAT1 (red signal) in EV and NS4B transfected cells; 6–10 fields per each condition, from a total of 3 biologically independent experiments, were analyzed; cells count was performed using Cell Counter plugin of ImageJ. **** p < 0.0001 values are reported, as obtained by unpaired t test.

Article Snippet: Membranes were probed with monoclonal rabbit primary antibodies against P-STAT1 (1:1000) and GAPDH (1:1000) (Cell Signaling, Denver, MA, USA), and polyclonal rabbit primary antibody against STAT1-tot (1:1000) (Cell Signaling, Denver, MA, USA).

Techniques: Immunofluorescence, Transfection, Plasmid Preparation, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: IFN-alpha-induced signal transduction, gene expression, and antitumor activity of immune effector cells are negatively regulated by suppressor of cytokine signaling proteins.

doi: 10.4049/jimmunol.178.8.4832

Figure Lengend Snippet: FIGURE 4. Overexpression of SOCS1 and SOCS3 protein in Jurkat cells inhibits the response to IFN-. The Jurkat T cell lymphoma cell line was transduced with PINCO retroviral constructs encoding SOCS1, SOCS2, or SOCS3 protein. Cells transduced with the empty PINCO vector served as a negative control. Cell populations were routinely 95% pure post-FACS for GFP. A, Transcript levels of SOCS1, SOCS2, and SOCS3 in transduced cells as measured by real-time PCR. Data are expressed as the mean fold increase relative to baseline levels. All real-time PCR data were normalized to the level of -actin mRNA in duplicate experiments. B, Protein levels of SOCS1, SOCS2, and SOCS3 as measured by immunoblot analysis of whole cell lysates. These results are representative of duplicate experiments. C, Flow cytometric analysis of P-STAT1 formation in transduced cells following stimulation with

Article Snippet: A rabbit anti-P-STAT1 (Tyr701) primary Ab (Cell Signaling Technology) was used in combination with a goat anti-rabbit Alexa Fluor 488-conjugated secondary Ab (Molecular Probes).

Techniques: Over Expression, Transduction, Retroviral, Construct, Plasmid Preparation, Negative Control, Real-time Polymerase Chain Reaction, Western Blot