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: 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

Journal: eLife

Article Title: Tuberculosis-associated IFN-I induces Siglec-1 on tunneling nanotubes and favors HIV-1 spread in macrophages

doi: 10.7554/eLife.52535

Figure Lengend Snippet:

Article Snippet: Antibody , Rabbit monoclonal anti‐human STAT1 (clone 42H3) , Cell Signaling Technology , Cat# 9175 RRID: AB_2197984 , WB (1:100).

Techniques: Derivative Assay, Cell Culture, Transfection, Construct, Enzyme-linked Immunosorbent Assay, Blocking Assay, Control, Recombinant, Isolation, Western Blot, Fluorescence, Software, Imaging

Journal: iScience

Article Title: RGS2 is an innate immune checkpoint for suppressing Gαq-mediated IFNγ generation and lung injury

doi: 10.1016/j.isci.2025.111878

Figure Lengend Snippet:

Article Snippet: Rabbit Monoclonal anti-phospho-STAT1 (Y701) , Cell Signaling , Cat # 9177S.

Techniques: Recombinant, Protease Inhibitor, Red Blood Cell Lysis, Construct, Control, Software

Journal: Cell Communication and Signaling : CCS

Article Title: An inhibitory effect on the nuclear accumulation of phospho-STAT1 by its unphosphorylated form

doi: 10.1186/s12964-022-00841-3

Figure Lengend Snippet: A , B Cytoplasmic retention of Flag-tagged WT STAT1 in IFNγ-stimulated cells co-expressing R602L/Y701F-GFP. Transfected STAT1-negative U3A and HeLa cells expressing R602L/Y701F-GFP and WT-Flag were treated with 50 ng/ml IFNγ for 45 min and subsequently stained with an anti-Flag antibody. A The fluorescence micrographs show the intracellular distribution of Flag-tagged STAT1 co-expressed in cells transfected with R602L/Y701F-GFP as well as the localization of the corresponding Hoechst-stained nuclei (n = 3 independent transfections). B The histogram demonstrates the nucleocytoplasmic STAT1-Flag distribution in the absence and presence of R602L/Y701F-GFP in IFNγ-treated HeLa cells, as determined by the ratio of nuclear-to-total fluorescence intensity (means ± standard deviations from n = 20 cells, * p ≤ 0.05, as assessed by a two-tailed Student’s t test). C The absence of nuclear P-STAT1 staining of the endogenous protein in HeLa cells expressing R602L/Y701F-GFP. HeLa cells were transfected with R602L/Y701F-GFP and stimulated with IFNγ for indicated times before staining with an anti-phospho-tyrosine antibody. D Crystal structure of the STAT1 anti-parallel dimer. A critical arginine residue at position 274 marked in cyan in the coiled-coil domains and threonine residue at position 385 marked in pink in the DNA-binding domains of the two STAT1 proteins (white and orange). Structural data were obtained from the Protein Data Bank (pdb) file 1YVL for the STAT1 anti-parallel dimer. E The absence of nuclear P-STAT1 staining of endogenous STAT1 in HeLa cells expressing R274W/R602L/Y701F-GFP. HeLa cells were transfected with R274W/R602L/Y701F-GFP and stimulated with IFNγ for indicated times before being stained with an anti-phospho-tyrosine antibody. Scale bars in A , C , E mark a distance of 10 µm

Article Snippet: The membranes were incubated with either a rabbit monoclonal phospho-Tyr701-specific STAT1 antibody (Cell Signaling Technology, 58D6) or monoclonal pan-STAT1 antibody (Cell Signaling Technology, D1K9Y) overnight.

Techniques: Expressing, Transfection, Staining, Fluorescence, Two Tailed Test, Residue, Binding Assay

Journal: Cell Communication and Signaling : CCS

Article Title: An inhibitory effect on the nuclear accumulation of phospho-STAT1 by its unphosphorylated form

doi: 10.1186/s12964-022-00841-3

Figure Lengend Snippet: The dimerization-deficient quadruple mutant (QM-GFP) mimicking U-STAT1 inhibits the detection of co-expressed WT protein but does not affect tyrosine phosphorylation. A – C HeLa cells were transfected with a plasmid coding for QM-GFP and stimulated with IFNγ for 45 min followed by staining with anti-phospho-tyrosine antibody. A The fluorescence micrographs show the intracellular distribution of QM-GFP and WT P-STAT1, as well as the localization of the corresponding Hoechst-stained nuclei. B Histogram demonstrating the net reduction of nuclear P-STAT1 intensity in HeLa cells expressing QM-GFP, as determined by the ratio of nuclear-to-total fluorescence intensity (n = 3 independent transfections, means ± standard deviations from n = 20 cells, * p ≤ 0.05 by a two-tailed Student’s t test). C Concentration-dependent inhibition of nuclear P-STAT1 accumulation by co-expressed QM-GFP. The fluorescence intensities of nuclear P-STAT1 staining were plotted against the total cellular QM-GFP fluorescence. D , E Representative immunoblot of whole cell extracts from STAT1-negative U3A cells co-expressing untagged STAT1 and QM-GFP after treatment for 45 min with 50 ng/ml of recombinant IFNγ and the quantification thereof from three independent transfection experiments. F , G An in vitro phosphorylation assay demonstrated no difference in tyrosine phosphorylation rates of the WT STAT1 by JAK2 with respect to the presence or absence of QM-GFP. Whole cell extracts (10 μl in each reaction) from reconstituted U3A cells expressing untagged STAT1 in combination with WT-GFP or QM-GFP were incubated with 4 μg/ml of recombinant JAK2 kinase and the levels of P-STAT1 were monitored over time by means of Western blotting (n = 3). Statistical analysis revealed no significant difference in the phosphorylation kinetics of the WT in the presence of either QM-GFP or WT-GFP. H , I Results from an in vitro dephosphorylation assay using extracts from IFNγ-pre-stimulated U3A cells expressing untagged STAT1 in combination with WT-GFP or QM-GFP (10 μl each) incubated for 0, 15 and 30 min with 2 U of the STAT-specific Tc45 phosphatase (n = 3). Tyrosine dephosphorylation was followed by immunoblotting including a quantitative analysis of the phospho-tyrosine signals divided by total amount of STAT1 signal. Scale bar in A marks a distance of 10 µm

Article Snippet: The membranes were incubated with either a rabbit monoclonal phospho-Tyr701-specific STAT1 antibody (Cell Signaling Technology, 58D6) or monoclonal pan-STAT1 antibody (Cell Signaling Technology, D1K9Y) overnight.

Techniques: Mutagenesis, Phospho-proteomics, Transfection, Plasmid Preparation, Staining, Fluorescence, Expressing, Two Tailed Test, Concentration Assay, Inhibition, Western Blot, Recombinant, In Vitro, Incubation, De-Phosphorylation Assay

Journal: Cell Communication and Signaling : CCS

Article Title: An inhibitory effect on the nuclear accumulation of phospho-STAT1 by its unphosphorylated form

doi: 10.1186/s12964-022-00841-3

Figure Lengend Snippet: Unaltered DNA binding, reporter activation and target gene induction by WT STAT1 in the presence of a dimerization-deficient mutant. A Electrophoretic mobility shift assay demonstrated unchanged binding of the recombinant WT protein from reconstituted U3A cell extracts to a [ 33 P]-radioactively labelled M67 probe containing a single GAS sequence, whereas the co-expressed unphosphorylated STAT1 variants and empty vector (pEGFPN1) showed no DNA-binding indicated by the absence of a GFP-tagged STAT1 band in lanes 3 to 5. The asterisk at the margin of the gelshift indicates an unspecific band. B EMSA result showing the unaltered capacity of endogenous STAT1 from HeLa cell extracts to form tetramers on the radioactively-labelled DNA probe 2xGAS, containing two GAS sites in tandem orientation, in the presence of co-expressed QM-GFP. HeLa cells were either untransfected (UT) or transfected with the indicated expression plasmids, and on the next day the cells were treated for 45 min with IFNγ or left untreated. Extracts were incubated with 2xGAS DNA probed for 15 min before subsequently being challenged by a 750-fold molar excess of unlabelled M67 DNA incubated for 10 min at RT in a competition assay (comp). For super-shift assays, 20 ng of STAT1- (αS1) or STAT3-specific (αS3) antibody were added to the reaction for 40 min at RT. C , D EMSA result showing the unaltered cytoplasmic and nuclear fractions of endogenous P-STAT1 from HeLa cell lysates bound to a [ 33 P]-radioactively labelled M67 probe containing a single GAS sequence, as the additional presence of QM-GFP did not hamper nuclear import of the endogenous STAT1 but its nuclear retention. E HeLa cells expressing WT-GFP, empty vector (pEGFPN1) or the quadruple mutant QM-GFP were left untreated (−) or stimulated for 6 h with 50 ng/ml of IFNγ (+). In whole extracts from these cells, luciferase luminescence of a reporter construct with a triple GAS site (3xLy6E) and the enzymatic activity of the co-expressed β-galactosidase were measured and represented graphically. F U3A cells were transfected with a STAT1-responsive luciferase reporter construct, a β-galactosidase expression vector and a combination of equal amounts of WT-GFP and the indicated STAT1 variants. These cells were either unstimulated (−) or stimulated with 50 ng/ml of IFNγ (+) and subsequently luciferase activity, normalized to the β-galactosidase expression, was measured in whole cell extracts and represented graphically. The experiment was repeated in six independent transfections at least three times. G – J U3A cells were untransfected (UT) or transfected with a plasmid coding for WT-GFP alone or a combination of WT-GFP and the indicated U-STAT1 variants or empty GFP vector (pEGFPN1). These cells where either untreated or stimulated with 50 ng/ml of IFNγ for 6 h. after which RNA was isolated and converted to cDNA. Histograms show the results from qPCR experiments for the following STAT1 target genes: G GBP1 , H MIG , I CXCL10 and J IRF1 . Gene induction was normalized to the expression of the house-keeping gene GAPDH . Histograms show means and standard deviations wherein the significant differences between the IFNγ-stimulated variant samples and cells expressing a single transfection of WT protein are marked by bars and asterisks. The experiment was repeated three times

Article Snippet: The membranes were incubated with either a rabbit monoclonal phospho-Tyr701-specific STAT1 antibody (Cell Signaling Technology, 58D6) or monoclonal pan-STAT1 antibody (Cell Signaling Technology, D1K9Y) overnight.

Techniques: Binding Assay, Activation Assay, Mutagenesis, Electrophoretic Mobility Shift Assay, Recombinant, Sequencing, Plasmid Preparation, Transfection, Expressing, Incubation, Competitive Binding Assay, Luciferase, Construct, Activity Assay, Isolation, Variant Assay

Journal: Cell Communication and Signaling : CCS

Article Title: An inhibitory effect on the nuclear accumulation of phospho-STAT1 by its unphosphorylated form

doi: 10.1186/s12964-022-00841-3

Figure Lengend Snippet: Deletion of the amino-terminus in mutant U-STAT1 restores nuclear accumulation of co-expressed, endogenous P-STAT1. A , B HeLa cells were transfected with an expression plasmid coding for R602L/Y701F-GFP, QM-GFP, ΔN/R602L/Y701F-GFP and QM/L407A/L409A-GFP and stimulated for 0 min or 45 min with 50 ng/ml of recombinant IFNγ, followed by staining with an anti-phospho-tyrosine antibody. Fluorescence micrographs show restored nuclear accumulation of P-STAT1 in the presence of amino-terminal deletion mutant ΔN/R602L/Y701F-GFP and the dsNLS mutant QM/L407A/L409A-GFP. Histograms show the quantification of nuclear P-STAT1 from HeLa cells co-expressing the indicated STAT1 variants in comparison with untransfected HeLa cells from three independent experiments (n = 3, means ± standard deviations from n = 20 cells, * p ≤ 0.05). C Fluorescence micrographs displaying the absence of nuclear endogenous P-STAT1 staining in the presence of mutant U-STAT1 constructs expressed in HeLa cells stimulated for 0 min and 45 min with recombinant IFNα (50 ng/ml). Note the rescue effect mediated by the deletion of the amino-terminus in the construct ΔN/R602L/Y701F-GFP under stimulation with type I IFN. Scale bars in A , C mark a distance of 10 µm. D Cellular fractionation experiments using cytoplasmic and nuclear lysates from HeLa cells expressing the indicated STAT1 variants demonstrate the presence of endogenous phospho-STAT1 in both compartments in IFNγ-pretreated cells. Representative immunoblots demonstrate unchanged tyrosine phosphorylation of endogenous STAT3 in whole cell extracts from HeLa cells expressing U-STAT1, after stimulation with IFNγ (lower panels). E , F Quantification from immunoblots of whole extracts from HeLa cells expressing the indicated mutants, including the quadruple mutant (QM), before and after treatment for 45 min with 50 ng/ml of recombinant IFNγ from three independent transfection experiments for the expression of phospho-STAT1 ( E ) and the expression of phospho-STAT3 ( F )

Article Snippet: The membranes were incubated with either a rabbit monoclonal phospho-Tyr701-specific STAT1 antibody (Cell Signaling Technology, 58D6) or monoclonal pan-STAT1 antibody (Cell Signaling Technology, D1K9Y) overnight.

Techniques: Mutagenesis, Transfection, Expressing, Plasmid Preparation, Recombinant, Staining, Fluorescence, Comparison, Construct, Cell Fractionation, Western Blot, Phospho-proteomics

Journal: Cell Communication and Signaling : CCS

Article Title: An inhibitory effect on the nuclear accumulation of phospho-STAT1 by its unphosphorylated form

doi: 10.1186/s12964-022-00841-3

Figure Lengend Snippet: Proposed model of U-STAT1-dependent import modulation of P-STAT1. The left panel describes a condition of low-level cellular U-STAT1, wherein fully functional import complexes of P-STAT1 are formed and translocated to the nucleus in cytokine-stimulated cells. The right panel depicts a high concentration of U-STAT1, wherein U-STAT1 interacts through its amino-terminus and dsNLS with both components of the importin dimer. The formation for a full-fledged import complex is hindered by the missing tyrosine phosphorylation residue of U-STAT1

Article Snippet: The membranes were incubated with either a rabbit monoclonal phospho-Tyr701-specific STAT1 antibody (Cell Signaling Technology, 58D6) or monoclonal pan-STAT1 antibody (Cell Signaling Technology, D1K9Y) overnight.

Techniques: Functional Assay, Concentration Assay, Phospho-proteomics, Residue