Journal: medRxiv

Article Title: Polymorphism in IFNAR contributes to glucocorticoid response and outcome in ARDS and COVID-19

doi: 10.1101/2022.03.10.22272123

Figure Lengend Snippet: (A ) STAT1 expression in the lung after 4-day culture in the presence of IFN beta with or without hydrocortisone (HC). ( B) pSTAT1 expression in the same specimens as in A. ( C) Example photomicrographs showing higher STAT2 expression in a TT patient than in a CT patient and the effect of HC on its nuclear translocation. Most STAT2 remains in the cytoplasm of the CT patients, whereas nuclear expression is prominent in the TT patient. Indicated insets are shown in the bottom row. Arrows. ( D ) Combined results of all patients noting that two CT samples are excluded in the data as the patients were already under glucocorticoid treatment at the time of sample acquisition. Ns, not significant; *P<0.05; **P<0.01; and ***P<0.001

Article Snippet: The first stage antibodies were anti-alpha chain of the IFN alpha/beta receptor (St John’s Laboratory STJ112765) that was used 1:2000 and 1:5000 and anti-Stat1 (1:400, 9175S), anti-pStat1(1:100, 9167S) and anti-Stat2 (1:200, 72604S) all from Cell Signalling.

Techniques: Expressing, Translocation Assay

Journal: The Journal of Biological Chemistry

Article Title: Inhibitor of ?B Kinase ? (IKK?), STAT1, and IFIT2 Proteins Define Novel Innate Immune Effector Pathway against West Nile Virus Infection *

doi: 10.1074/jbc.M111.285205

Figure Lengend Snippet: Virus infection induces delayed STAT1 Ser-708 phosphorylation. A, WT or IKKϵ−/− MEFs were mock-stimulated or stimulated with 100IU/ml IFN-β. Protein lysate was collected 16 h post-IFN stimulation and immunoblotted using p-STAT1 Ser-708, p-STAT1 Ser-727, p-STAT1 Tyr-701, and total STAT1 antibodies. B and C, Sendai and WNV virus infections induce STAT1 Ser-708 phosphorylation. HEK293 cells were infected with 100 HA units/ml SenV (B) or West Nile virus strain Madagascar (WNV-MAD) (C) at an m.o.i. of 1. At the indicated times following infection, protein lysates were collected and immunoblotted for p-STAT1 Ser-708, p-STAT1 Tyr-701, total STAT1, p-IRF-3, total IRF-3, IFIT1, and SenV or WNV. Tubulin and GAPDH were used as loading controls.

Article Snippet: Affinity-purified rabbit polyclonal anti-STAT1 Ser-708 antibody was generated by repeat-immunization with the STAT1 Ser-708 phospho-specific peptide (YIKTELI{pS}VSEVHP, amino acids 701–714) (GenScript).

Techniques: Infection

Journal: The Journal of Biological Chemistry

Article Title: Inhibitor of ?B Kinase ? (IKK?), STAT1, and IFIT2 Proteins Define Novel Innate Immune Effector Pathway against West Nile Virus Infection *

doi: 10.1074/jbc.M111.285205

Figure Lengend Snippet: Type I, type II, and type III IFNs induce STAT1 Ser-708 phosphorylation. 2fTGH cells were mock-treated or treated with 100 IU/ml IFN-β (A) or 50 ng/ml IFN-γ (B). C, PH5CH8 cells were mock-treated (lane 1), treated with 100 ng/ml IFN-λ1 (lanes 2-6), or 100 IU/ml IFN-β (lanes 7-9). Protein lysate was collected at respective time points following IFN treatment and immunoblotted for p-STAT1 Ser-708, p-STAT1 Tyr-701, p-STAT1 Ser-727, total STAT1, ADAR1, and IFIT1.

Article Snippet: Affinity-purified rabbit polyclonal anti-STAT1 Ser-708 antibody was generated by repeat-immunization with the STAT1 Ser-708 phospho-specific peptide (YIKTELI{pS}VSEVHP, amino acids 701–714) (GenScript).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Inhibitor of ?B Kinase ? (IKK?), STAT1, and IFIT2 Proteins Define Novel Innate Immune Effector Pathway against West Nile Virus Infection *

doi: 10.1074/jbc.M111.285205

Figure Lengend Snippet: Signaling through IFNAR is required for STAT1 Ser-708 phosphorylation following type I IFN treatment or virus infection. WT, IRF-3−/−, or IFNAR−/− (A) and parental 2fTGH cells or their derivative U5A cells (which lack IFNAR) (B) were infected with WNV-MAD at an m.o.i. of 1. Protein lysates were collected at the indicated time points and immunoblotted for p-STAT1 Ser-708, p-STAT1 Tyr-701, p-STAT1 Ser-727, total STAT1, p-IRF-3, total IRF-3, and WNV. C and D, the same cells were also mock-stimulated or stimulated with 100 IU/ml IFN-β for 6 or 16 h. Protein lysates were collected at the indicated time points and immunoblotted for p-STAT1 Ser-708, p-STAT1 Tyr-701, p-STAT1 Ser-727, total STAT1, p-IRF-3, total IRF-3, IFIT2, IFIT3, and IFIT1. Asterisk, nonspecific band.

Article Snippet: Affinity-purified rabbit polyclonal anti-STAT1 Ser-708 antibody was generated by repeat-immunization with the STAT1 Ser-708 phospho-specific peptide (YIKTELI{pS}VSEVHP, amino acids 701–714) (GenScript).

Techniques: Infection

Journal: The Journal of Biological Chemistry

Article Title: Inhibitor of ?B Kinase ? (IKK?), STAT1, and IFIT2 Proteins Define Novel Innate Immune Effector Pathway against West Nile Virus Infection *

doi: 10.1074/jbc.M111.285205

Figure Lengend Snippet: STAT1 Ser-708 phosphorylation requires de novo protein synthesis, STAT1 tyrosine dephosphorylation, and nuclear export. A, 2fTGH cells were mock-treated (-CHX, lanes 1-5) or treated with CHX (+CHX, lanes 6-11) to block protein synthesis. At 30 min (lanes 6-10) or 16 h (lane 11) following CHX treatment, cells were mock-stimulated (M) or stimulated with IFN-β. Cells were harvested at 10 min as well as 1, 6, and 16 h post-IFN stimulation and immunoblotted to detect p-STAT1 Ser-708, p-STAT1 Tyr-701, total STAT1, ISG15, and IFIT1. B, 2fTGH cells were not treated (NT, lanes 1-3), pretreated with 100 nm LMB (lanes 4-6), or 50 mm pervanadate (Van, lanes 7-9) 1 h before mock stimulation or stimulation with 100 IU/ml IFN-β. Cells were harvested at 1 and 16 h post-stimulation. Immunoblot analysis was performed using p-STAT1 Ser-708, p-STAT1 Tyr-701, p-STAT1 Ser-727, total STAT1, ADAR1, and IFIT1 antibodies.

Article Snippet: Affinity-purified rabbit polyclonal anti-STAT1 Ser-708 antibody was generated by repeat-immunization with the STAT1 Ser-708 phospho-specific peptide (YIKTELI{pS}VSEVHP, amino acids 701–714) (GenScript).

Techniques: De-Phosphorylation Assay, Blocking Assay, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Inhibitor of ?B Kinase ? (IKK?), STAT1, and IFIT2 Proteins Define Novel Innate Immune Effector Pathway against West Nile Virus Infection *

doi: 10.1074/jbc.M111.285205

Figure Lengend Snippet: IKKϵ mediates IFIT2 expression and protection against WNV pathogenesis in vivo. WT Bl6 and IKKϵ−/− mice were mock-infected (PBS only) or infected with 103 pfu of WNV-MAD subcutaneously through footpad injection. A, mice were monitored and scored daily for clinical symptoms over 17 days. Clinical scores from four representative mice per group were graphed. B, spleens from WT or IKKϵ−/− mice, mock-infected or infected with WNV-MAD, were collected at days 4, 6, and 12 post-infection. Protein lysates were extracted by homogenizing spleens with radioimmune precipitation assay buffer and immunoblotted using p-STAT1 Ser-708, p-STAT1 Tyr-701, p-STAT1 Ser-727, total STAT1, IFIT2, IFIT1, WNV, and IKKϵ antibodies. The immunoblot analysis panel is a representative from four mice per infection group.

Article Snippet: Affinity-purified rabbit polyclonal anti-STAT1 Ser-708 antibody was generated by repeat-immunization with the STAT1 Ser-708 phospho-specific peptide (YIKTELI{pS}VSEVHP, amino acids 701–714) (GenScript).

Techniques: Expressing, In Vivo, Infection, Injection, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Inhibitor of ?B Kinase ? (IKK?), STAT1, and IFIT2 Proteins Define Novel Innate Immune Effector Pathway against West Nile Virus Infection *

doi: 10.1074/jbc.M111.285205

Figure Lengend Snippet: A model illustrating that early and late ISGs induction is regulated by multiple STAT1 posttranslational modifications. 1, the canonical JAK-STAT signaling is activated following type I IFN binding to its receptor, which results in STAT1 Tyr-701 phosphorylation, ISGF3 formation, and its nuclear translocation. ISGF3 binding to the ISRE element induces transcription of ISGs. 2, chromatin-bound STAT1 can be phosphorylated by MAPK at residue Ser-727, which induces its sumoylation. 3 and 4, nuclear STAT1 is also acetylated by histone acetyltransferase (HAT) CREB-binding protein (CBP), resulting in recruitment of TCP1, which catalyzes STAT1 tyrosine dephosphorylation. Sumoylated-acetylated STAT1 cycles back to the cytoplasm, and both modifications render STAT1 unable to be further tyrosine-phosphorylated. 5 and 6, type I IFN signaling and unknown IFN-stimulated factor(s) activate IKKϵ phosphorylation of STAT1 Ser-708. 7, STAT1 molecules phosphorylated at Ser-708 can enter the nucleus and induce expression of a specific ISG subset. pY, tyrosine phosphorylation; pS, serine phosphorylation; Ac, acetylation; Su, sumoylation.

Article Snippet: Affinity-purified rabbit polyclonal anti-STAT1 Ser-708 antibody was generated by repeat-immunization with the STAT1 Ser-708 phospho-specific peptide (YIKTELI{pS}VSEVHP, amino acids 701–714) (GenScript).

Techniques: Binding Assay, Translocation Assay, De-Phosphorylation Assay, Expressing

Journal: medRxiv

Article Title: Neonatal-onset autoinflammation and immunodeficiency caused by heterozygous missense mutation of the proteasome subunit β-type 9

doi: 10.1101/2021.02.01.21250077

Figure Lengend Snippet: Panels A and B show the proteasome analysis of unstimulated or IFN-γ-stimulated skin fibroblasts from a healthy control and patient 2. Cell extracts were prepared and fractionated by glycerol gradient centrifugation. Panel A shows immunoblot analysis of each fraction using Abs against the indicated proteins. Panel B shows chymotrypsin-like activity of each fraction, which was measured by using Suc-LLVY-AMC as a substrate in the absence (top) or presence (bottom) of 0.0025% SDS. Panel C shows the STAT1 phosphorylation assay with SV40-transformed dermal fibroblasts of control subjects and Patient 2. The expression levels of IFN-γ induced p-STAT1 were enhanced in patient 2 compared to control subjects. Panel D shows the skin biopsy sample of patient 2 stained with anti-ubiquitin Ab. A scale bar represents 100 μm.

Article Snippet: Six μm sections of biopsy samples from the patients’ skin lesion were stained with rabbit polyclonal anti-p-STAT1 Abs (GeneTex) and anti-ubiquitin Abs (DAKO).

Techniques: Control, Gradient Centrifugation, Western Blot, Activity Assay, Phospho-proteomics, Transformation Assay, Expressing, Staining, Ubiquitin Proteomics

Journal: Bioorganic chemistry

Article Title: Bromo-substituted indirubins for inhibition of protein kinase-mediated signalling involved in inflammatory mediator release in human monocytes.

doi: 10.1016/j.bioorg.2024.107470

Figure Lengend Snippet: Fig. 3. Impact of indirubins 1 and 2 on the activation status of protein kinase pathways. A-F) Human monocytes were pre-treated with indirubins 1 or 2 at the indicated concentrations, or with inhibitors of GSK-3β (SB216763, 5 µM), CDK8 (CCT-251921, 40 nM), CDK9 (JSH-150, 20 nM) or CDK1 (Ro-3306, 10 µM) for 15 min prior to stimulation with LPS 100 ng/mL for 18 h. (A) Protein expression of β-catenin compared to GAPDH as housekeeping protein. (B) Levels of phosphorylated GSK-3β compared to total expression of GSK-3β protein. (C,D) Levels of phosphorylated STAT1 (C, Ser727 and D, Tyr701) vs. β-actin. (E) Levels of phosphorylated NF-κB (Ser276) compared to GAPDH. (F) Levels of phosphorylated Rb (Ser807/Ser811) vs. β-actin. Protein amounts were evaluated by Western Blot and densito metric analysis thereof. n = 3–4 biological replicates. Statistics: Data are shown as A-E) individual values and means ± SEM. Statistics were calculated by one-way ANOVA for multiple comparisons with Dunnett’s correction, testing treatments against vehicle (0.1 % (v/v) DMSO). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant.

Article Snippet: Membranes were incubated with the following primary antibodies: rabbit monoclonal anti-COX-2, 1:1000 (12282, Cell Signaling, Danvers, MA), mouse monoclonal anti-β-catenin, 1:1000 (610153, BD Biosciences, San Jose, CA) mouse monoclonal anti-GSK-3β, 1:1000 (9832, Cell Signaling), rabbit monoclonal anti-phospho-GSK-3β (Ser9), 1:1000 (9323S, Cell Signaling), rabbit monoclonal anti-phosphoSTAT1 (Ser727), 1:1000 (ab109461, Abcam, Cambridge, UK), rabbit monoclonal anti-phospho-STAT1 (Tyr701), 1:1000 (ab109457, Abcam), rabbit monoclonal anti-phospho-NF-κB p65 (Ser276), 1:1000 (ab183559, Abcam), rabbit monoclonal anti-phospho-Rb (Ser807/811), 1:1000 (9308S, Cell Signaling), monoclonal anti-β-actin, 1:1000 (4970, Cell Signaling), mouse monoclonal anti-GAPDH, 1:1000 (97166S, Cell Signaling).

Techniques: Activation Assay, Expressing, Western Blot

Journal: Cell reports

Article Title: Tumor-specific CD4 T cells instruct monocyte fate in pancreatic ductal adenocarcinoma

doi: 10.1016/j.celrep.2023.112732

Figure Lengend Snippet: (A) NicheNet analysis from scRNA-seq data from using CD4 T cells as donor cells and MHCII hi macrophages as acceptor cells. See also . (B) Stat1 staining and MFI in MHCII hi and MHCII lo intratumoral macrophages at day 7. Data are mean ± SEM; n = 4 mice per group; ****p < 0.0001; Student’s t test. (C) Tumor weights from WT or Ifngr1 −/− mice; n = 4–8 mice per group; ****p < 0.0001; Student’s t test for each time point. (D) Proportion of MHCII hi or MHCII lo macrophages from WT or Ifngr1 −/− mice. Data are mean ± SEM; n = 4–8 mice per group; ****p < 0.0001; Student’s t test for each time point. (E) Proportion of Arg1 + macrophages on day 7. Data are mean ± SEM; ****p < 0.0001; Student’s t test. (F) Proportion of MHCII lo CD206 + FRβ + macrophages from WT or Ifngr1 −/− mice. Data are mean ± SEM; n = 4–8 mice per group; ****p < 0.0001; Student’s t test for each time point. (G) PD-L1 MFI gated on total macrophages from WT or Ifngr1 −/− mice. Data are mean ± SEM; **p < 0.005; Student’s t test for each time point. (H) Normalized expression of selected genes from bulk RNA sequencing from sorted live MHCII hi or MHCII lo tumor macrophages from WT and Ifngr1 −/− mice at day 14. See also . (I) Gene set enrichment analysis (GSEA) of the top 300 differentially expressed genes from sorted cells in (H). (J) Orthotopic KPC 2a tumor weights at day 7 from WT control mice, WT mice treated with CD40L blockade (αCD40L), or Cd40 −/− mice. *p < 0.05; one way ANOVA with Tukey’s post test. See also . (K) Proportion of MHCII hi or MHCII lo macrophages. Each dot is an independent mouse. Data are mean ± SEM; n = 4–5 mice per group; ***p < 0.001; one-way ANOVA with Tukey’s posttest. (L) Proportion of Arg1 + macrophages. Each dot is an independent mouse. Data are mean ± SEM; n = 4–5 mice per group; ***p < 0.001; one-way ANOVA with Tukey’s posttest. (M) Proportion of MHCII lo CD206 + FRβ + macrophages. Each dot is an independent mouse. Data are mean ± SEM; n = 4–5 mice per group; ***p < 0.001; one-way ANOVA with Tukey’s posttest. See also .

Article Snippet: anti-mouse Stat1 (clone D1K9Y) , Cell Signaling , Cat#: 80916S.

Techniques: Staining, Expressing, RNA Sequencing, Control

Journal: Cell reports

Article Title: Tumor-specific CD4 T cells instruct monocyte fate in pancreatic ductal adenocarcinoma

doi: 10.1016/j.celrep.2023.112732

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: anti-mouse Stat1 (clone D1K9Y) , Cell Signaling , Cat#: 80916S.

Techniques: Recombinant, Red Blood Cell Lysis, Cell Isolation, Cell Stimulation, Software