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: Journal of Inflammation Research

Article Title: Huiyang Shengji Unguent Promotes Lymphangiogenesis and Wound Healing in Diabetic Chronic Wounds: Combined Insights from Proteomics and in vivo and in vitro Analyses

doi: 10.2147/JIR.S534105

Figure Lengend Snippet: HYSJ inhibits activation of the pyroptosis pathway in HLECs in a high glucose inflammation model. ( A ) Western blot analysis of upstream proteins involved in pyroptosis, including TLR2, Myd88, and Stat1, in HLECs under the indicated conditions. ( B – D ) Relative expression level analysis of TLR2, Myd88, and Stat1 proteins. ( E ) Pyroptosis pathway proteins NLRP3, caspase-1, gasdermin D, and N-gasdermin D expression. ( F – I ) Relative expression of pyroptosis pathway proteins NLRP3, caspase-1, gasdermin D, and Ngasdermin D. ( J ) Immunofluorescence detection of N-gasdermin D expression in HLECs under the indicated intervention conditions. ( K ) Fluorescence intensity analysis of Ngasdermin D in each group. ( L ) LDH levels in supernatants of HLECs under the indicated intervention conditions. Each group n=3. *P < 0.05, **P < 0.01, ***P<0.001, ****P < 0.0001 compared to model group.

Article Snippet: The membrane was incubated with the following primary antibodies as required for 10 hours at 4 °C:recombinant anti-NLRP3 antibody (1:1000; Cat. No. 4691S, Abcam, Cambridge, UK), caspase-1 (E9R2D) rabbit mAb (1:1000; Cat. No. 83383; Cell Signaling Technology, Boston, USA), gasdermin D (E9S1X) rabbit mAb (1:500; Cat. No. HA721144; HUAbio, Hangzhou, China), recombinant anti-LYVE1 antibody RM1067 (1:1000; Cat. No. ab314241; Abcam), PROX1 recombinant rabbit monoclonal antibody (1:500; Cat. No.HA722318; HUAbio), Flt-4 rabbit polyclonal antibody (1:1000; Cat. No. ER65750; HUAbio), VEGFC rabbit polyclonal antibody (1:1000; Cat. No. 22601–1; Proteintech, Wuhan, China), STAT1 recombinant rabbit monoclonal antibody (1:1000; Cat. No. ET1612; HUAbio), MyD88 rabbit pAb (1:1000; Cat. No. A0980; Abclonal, Wuhan, China), TLR2 recombinant rabbit monoclonal antibody (1:800; Cat. No. ET1705; HUAbio), or β-actin antibody (1:8000; Cat. No. #52901; Signalway Antibody, Maryland, USA).

Techniques: Activation Assay, Western Blot, Expressing, Immunofluorescence, Fluorescence

Journal: PLOS Pathogens

Article Title: IL-2–mTORC1 signaling coordinates the STAT1/T-bet axis to ensure Th1 cell differentiation and anti-bacterial immune response in fish

doi: 10.1371/journal.ppat.1010913

Figure Lengend Snippet: ( A ) Spleen leukocytes were stimulated with PHA for 12 h. Relative mRNA levels of T-bet, STAT1 and STAT4 were examined by qPCR, n = 4. ( B ) Flow cytometry showed the phosphorylation level of STAT1 in lymphocytes that stimulated with PHA. ( C ) Transcriptional levels of IFN-γ in HEK 293T cells that transfected with tilapia T-bet, STAT1 and STAT4, n = 6. ( D ) HEK 293T cells were co-transfected with tilapia T-bet, STAT1 or STAT4, and pGL3-IFN-γ promoter. The LUC activities were assessed at 48 h post-transfection, n = 4. ( E-G ) Spleen leukocytes from tilapia that i . p . injected with T-bet-specific, STAT1-specific or control siRNA for 2 days were harvested and stimulated with CD3ε mAb for 12 h. Relative mRNA levels of STAT1, T-bet and IFN-γ ( E, F , n = 4), and the percentage of CD3 + CD4-1 + IFN-γ + T cells ( G ) were examined. ( H-L ) Tilapia i . p . injected with T-bet-specific, STAT1-specific or control siRNA were infected with E . piscicida . Tilapia was i . p . injected with BFA 6 h before sacrifice, and spleen leukocytes were harvest for assay. Western blot assay showed the expression of IFN-γ at 48 h post infection ( H ). Flow cytometry and scatter plot figures showed the percentage and absolute numbers of CD3 + CD4-1 + T cells (I, K) and CD3 + CD4-1 + IFN-γ + T cells (J, L) on 5 DPI, n = 4. ( M-O ) Tilapia individuals that infected with E . piscicida were injected with STAT1 inhibitor Fludarabine or PBS, and animals were i . p . injected with BFA 6 hours before sacrifice. ( M ) Western blot assay showed the protein levels of IFN-γ in spleen leukocytes on indicated days. ( N ) Flow cytometry showed the percentage of IFN-γ in spleen CD4-1 + T cells on day 7 post-infection. ( O ) Kaplan-Meyer survival plot showed the survival percentage of tilapia, n = 25. These experiments were repeated for at least two independent times. *: p <0.05, **: p <0.01, ***: p <0.001, determined by a two-tailed Student’s t-test. The accession numbers of selected sequences were listed in .

Article Snippet: For p-STAT1 staining, leukocytes were fixed with Foxp3 Fixation/Permeabilization working solution (eBioscience) on ice for 2 h, and then stained with Alexa Fluor 647 conjugated-p-STAT1 (Tyr701) Rabbit mAb (CST) on ice for 30 min. Stained cells were then washed twice with 1× permeabilization buffer.

Techniques: Flow Cytometry, Phospho-proteomics, Transfection, Injection, Control, Infection, Western Blot, Expressing, Two Tailed Test

Journal: PLOS Pathogens

Article Title: IL-2–mTORC1 signaling coordinates the STAT1/T-bet axis to ensure Th1 cell differentiation and anti-bacterial immune response in fish

doi: 10.1371/journal.ppat.1010913

Figure Lengend Snippet: ( A, B ) Spleen leukocytes were stimulated with PHA or CD3ε/CD28 mAbs for 12 h, and mRNA levels of IFNγR1 and IFNγR2 were examined by qPCR, n = 6. ( C ) SDS-PAGE assay showed the recombination of tilapia IFNγR1 and IFNγR2 with GST-tag in E . coli . ( D ) GST pull-down assay showed the interaction of tilapia IFN-γ with IFNγR1 and IFNγR2. ( E ) Flow cytometry showed the phosphorylation level of STAT1 in lymphocytes that stimulated with recombinant IFN-γ. ( F, G ) Spleen leukocytes were stimulated with recombinant IFN-γ for 12 h, and mRNA levels of indicated molecules were examined by qPCR, n = 5. ( H ) Tilapia was injected with STAT1 inhibitor for 2 days before spleen leukocytes were stimulated with recombinant IFN-γ for 12 h. The expression levels of T-bet were examined by qPCR, n = 6. ( I-K ) Spleen leukocytes from tilapia that i . p . injected with T-bet-specific, STAT1-specific or control siRNA for 2 days were harvested and stimulated with recombinant IFN-γ for 12 h. Relative mRNA levels of STAT1, T-bet and IFN-γ ( I, J , n = 5), and the percentage of IFN-γ + cells in gated CD3 + CD4-1 + T cells ( K ) were examined. These experiments were repeated for at least two independent times. *: p <0.05, **: p <0.01, ***: p <0.001, determined by a two-tailed Student’s t-test.

Article Snippet: For p-STAT1 staining, leukocytes were fixed with Foxp3 Fixation/Permeabilization working solution (eBioscience) on ice for 2 h, and then stained with Alexa Fluor 647 conjugated-p-STAT1 (Tyr701) Rabbit mAb (CST) on ice for 30 min. Stained cells were then washed twice with 1× permeabilization buffer.

Techniques: SDS Page, Pull Down Assay, Flow Cytometry, Phospho-proteomics, Recombinant, Injection, Expressing, Control, Two Tailed Test

Journal: PLOS Pathogens

Article Title: IL-2–mTORC1 signaling coordinates the STAT1/T-bet axis to ensure Th1 cell differentiation and anti-bacterial immune response in fish

doi: 10.1371/journal.ppat.1010913

Figure Lengend Snippet: IL-2-mTORC1 signaling coordinates STAT1/T-bet axis to ensure Th1 cell differentiation and anti-bacterial immune response in tilapia.

Article Snippet: For p-STAT1 staining, leukocytes were fixed with Foxp3 Fixation/Permeabilization working solution (eBioscience) on ice for 2 h, and then stained with Alexa Fluor 647 conjugated-p-STAT1 (Tyr701) Rabbit mAb (CST) on ice for 30 min. Stained cells were then washed twice with 1× permeabilization buffer.

Techniques: Cell Differentiation

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