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: International Journal of Biological Sciences

Article Title: Comprehensive Analyses Reveal Effects on Tumor Immune Infiltration and Immunotherapy Response of APOBEC Mutagenesis and Its Molecular Mechanisms in Esophageal Squamous Cell Carcinoma

doi: 10.7150/ijbs.83824

Figure Lengend Snippet: A3A 's role in predicting immunotherapy response. ( A ) Heatmap showing the log2 transformed fold change in tumors relative to that in normal tissues of several important immune checkpoints. The P value of Wilcoxon rank sum test represented the significance. ** indicating P < 0.01 of two-sided Wilcoxon rank sum test. ( B ) A3A mRNA levels comparison between patients with different anti-PD-L1 treatment response. ( C ) The Kaplan-Meier survival curves according to A3A mRNA levels in patients receiving anti-PD-L1 treatment. ( D ) The proportion of patients with different treatment response in high and low A3A groups. ( E ) Representative multiplexed immunofluorescent staining pictures showing the positive correlation and co-localization between A3A and PD-L1 (N = 19, tissues collected from Cohort 1). Scale bar, 100 μm. ( F ) Western blot analysis of PD-L1, total and p-STAT1 in KYSE30 with A3A OE or KO. ( G ) Image of the mouse tumors with or without A3A overexpression receiving anti-PD-1 treatment or IgG control treatment at the end of the experiment. ( H-I ) Statistical graph showing the weight of subcutaneous tumors ( H ) and tumor growth curves showing the tumor volume ( I ) among the four groups (N=5 per group). Data are shown as mean ± SEM. * indicating P < 0.05, ** indicating P < 0.01, *** indicating P < 0.001, **** indicating P < 0.0001, and NS, not significant of Student's t-test. ( J ) A proposed model for the regulatory mechanism of APOBEC mutagenesis in immunity and immunotherapy response in ESCC.

Article Snippet: Vinculin (ab219649), STAT1 (ab234400), γH2AX (phospho S139, ab26350) antibodies were from Abcam, while antibodies against cGAS (#79978), STING (#13647), phospho-STING (p-STING) (Ser366; #50907), IRF3 (#11904), phospho-IRF3 (p-IRF3) (Ser396; #29047), TBK1 (#3504), phospho-TBK1 (p-TBK1) (Ser172; #5483), and phospho-STAT1 (p-STAT1) (Tyr701; #7649) were from Cell Signaling Technology.

Techniques: Transformation Assay, Comparison, Staining, Western Blot, Over Expression, Control, Mutagenesis

Journal: Antioxidants

Article Title: ROS-Driven STAT1 S-Glutathionylation Sustains IFNγ Signaling and Pro-Inflammatory Microglial Polarization

doi: 10.3390/antiox14121395

Figure Lengend Snippet: IFNγ treatment induces STAT1 activation in BV2 cells. BV2 cells were exposed to 20 ng/mL of IFNγ at different time points and activation of STAT1 was evaluated through different experiments. ( A ) Total protein extracts of IFNγ-treated BV2 cells at different time points were analyzed by Western Blot with anti-phospho-Tyr701 STAT1 antibody, and after stripping, the same blot was analyzed with anti-STAT1 antibody. Densitometric analysis revealed a STAT1 phosphorylation upon IFNγ treatment, peaking at 15 min compared with untreated BV2 cells (CTR). ( B ) RT-qPCR of iNOS and COX2, two STAT1-dependent genes, revealed an increase in their mRNA levels after 18 and 24 h of IFNγ treatment in BV2 cells. Data are expressed as mean ± SD (n = 3). * p < 0.05, ** p < 0.01 vs. untreated cells (CTR). Single data points are shown as red dots.

Article Snippet: Membranes were blocked with 5% BSA or 5% fat dry milk in Tris-buffered saline with 0.1% Tween 20 (TBST) at room temperature for 1 h and then incubated with primary antibodies specific for anti-phospho-Tyr701 STAT1 (sc-136229 Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-STAT1 (sc-346, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Tubulin (Oncogene, CP06-100UG), and anti-iNOS (ab-15323, Abcam, Cambridge, UK).

Techniques: Activation Assay, Western Blot, Stripping Membranes, Phospho-proteomics, Quantitative RT-PCR

Journal: Antioxidants

Article Title: ROS-Driven STAT1 S-Glutathionylation Sustains IFNγ Signaling and Pro-Inflammatory Microglial Polarization

doi: 10.3390/antiox14121395

Figure Lengend Snippet: GEE pre-treatment counteracts IFNγ-dependent STAT1 activation in BV2 cells. ( A ) Total protein extracts from BV2 cells pre-treated overnight with GEE and subsequently exposed to 20 ng/mL IFNγ for 18 h were analyzed by Western Blot using a phospho-Tyr701 STAT1 antibody. The same blot was analyzed with anti-Tubulin antibody to check the amount of loaded proteins. Densitometric analysis showed decreased STAT1 phosphorylation in GEE pre-treated cells compared to CTR. * p < 0.05, ** p < 0.01 vs. untreated BV2 cells (CTR). # p < 0.05 vs. IFNγ-treated cells. ( B ) RT-qPCR analysis revealed that iNOS and COX2 mRNA levels were reduced in BV2 cells pre-treated overnight with GEE and then exposed to 20 ng/mL IFNγ for 24 h. Data are expressed as mean ± SD (n = 3). ** p < 0.01 vs. CTR, ## p < 0.01 vs. IFNγ-treated cells. Single data points are shown as red dots.

Article Snippet: Membranes were blocked with 5% BSA or 5% fat dry milk in Tris-buffered saline with 0.1% Tween 20 (TBST) at room temperature for 1 h and then incubated with primary antibodies specific for anti-phospho-Tyr701 STAT1 (sc-136229 Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-STAT1 (sc-346, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Tubulin (Oncogene, CP06-100UG), and anti-iNOS (ab-15323, Abcam, Cambridge, UK).

Techniques: Activation Assay, Western Blot, Phospho-proteomics, Quantitative RT-PCR

Journal: Antioxidants

Article Title: ROS-Driven STAT1 S-Glutathionylation Sustains IFNγ Signaling and Pro-Inflammatory Microglial Polarization

doi: 10.3390/antiox14121395

Figure Lengend Snippet: GEE pre-treatment inhibits IFNγ-induced STAT1 phosphorylation and nuclear translocation in BV2 cells. The cells were immunostained with phospho-Tyr701 STAT1 (red) and analyzed by confocal microscopy (lens 60×). Nuclei were stained with DAPI (cyan). The orthogonal sections (ZX and ZY) of merge image reveal that pTYR 701 STAT1 protein is inside the nuclei. Scale bars indicate 10 μm. Images are representative of three separate experiments.

Article Snippet: Membranes were blocked with 5% BSA or 5% fat dry milk in Tris-buffered saline with 0.1% Tween 20 (TBST) at room temperature for 1 h and then incubated with primary antibodies specific for anti-phospho-Tyr701 STAT1 (sc-136229 Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-STAT1 (sc-346, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Tubulin (Oncogene, CP06-100UG), and anti-iNOS (ab-15323, Abcam, Cambridge, UK).

Techniques: Phospho-proteomics, Translocation Assay, Confocal Microscopy, Staining

Journal: Antioxidants

Article Title: ROS-Driven STAT1 S-Glutathionylation Sustains IFNγ Signaling and Pro-Inflammatory Microglial Polarization

doi: 10.3390/antiox14121395

Figure Lengend Snippet: IFNγ treatment induces S-glutathionylation of STAT1. ( A ) Total protein extracts of BV2 cells left untreated (CTR) or treated with 20 ng/mL IFNγ for the indicated times were subjected to immunoprecipitation with anti-STAT1 antibody. Immunoprecipitated STAT1 (IP: STAT1) was analyzed by Western Blot under non-reducing conditions using anti-SSG antibody and, after membrane stripping, re-probed with anti-STAT1 antibody. Densitometric analysis revealed increased STAT1 S-glutathionylation after 5 and 15 min of IFNγ treatment compared to untreated BV2 cells. * p < 0.05, ** p < 0.01 vs. untreated BV2 cells (CTR). ( B ) Total protein lysates from the same untreated and treated BV2 cells were reserved before pull-down (input) and analyzed by Western Blot with anti-phospho-Tyr701 STAT1 antibody and, after membrane stripping, re-probed with anti-STAT1 antibody. Images are representative of four independent experiments. Single data points are shown as red dots.

Article Snippet: Membranes were blocked with 5% BSA or 5% fat dry milk in Tris-buffered saline with 0.1% Tween 20 (TBST) at room temperature for 1 h and then incubated with primary antibodies specific for anti-phospho-Tyr701 STAT1 (sc-136229 Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-STAT1 (sc-346, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Tubulin (Oncogene, CP06-100UG), and anti-iNOS (ab-15323, Abcam, Cambridge, UK).

Techniques: Immunoprecipitation, Western Blot, Membrane, Stripping Membranes

Journal: Antioxidants

Article Title: ROS-Driven STAT1 S-Glutathionylation Sustains IFNγ Signaling and Pro-Inflammatory Microglial Polarization

doi: 10.3390/antiox14121395

Figure Lengend Snippet: FNγ induces M1 polarization in BV2 cells through oxidative stress and STAT1 activation. ( A ) The expression of the pro-inflammatory cytokines IL-6 and TNFα in the supernatant of BV2 cells was evaluated by ELISA after 18 h of IFNγ stimulation, with or without GEE pretreatment. GEE markedly reduced the IFNγ-induced secretion of both cytokines, confirming the involvement of oxidative stress in M1 polarization. Data are expressed as mean ± SD (n = 3). * p < 0.05, ** p < 0.01 vs. CTR. # p < 0.05 vs. IFNγ. ( B ) Parental and STAT1 knockdown in BV2 cells were treated with 20 ng/mL of IFNγ for 18 h. Total protein extracts were analyzed by Western Blot with anti-iNOS and anti-Actin antibodies. Densitometric analysis revealed reduced iNOS induction in STAT1 knockdown in BV2 cells compared with parental cells upon IFNγ treatment. Parental and STAT1 knockdown in BV2 cells cultured without IFNγ were used as controls (CTR). Images are representative of three independent experiments. Single data points are shown as red dots. * p < 0.05, ** p < 0.01 vs. CTR. # p < 0.05 vs. IFNγ.

Article Snippet: Membranes were blocked with 5% BSA or 5% fat dry milk in Tris-buffered saline with 0.1% Tween 20 (TBST) at room temperature for 1 h and then incubated with primary antibodies specific for anti-phospho-Tyr701 STAT1 (sc-136229 Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-STAT1 (sc-346, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Tubulin (Oncogene, CP06-100UG), and anti-iNOS (ab-15323, Abcam, Cambridge, UK).

Techniques: Activation Assay, Expressing, Enzyme-linked Immunosorbent Assay, Knockdown, Western Blot, Cell Culture

Journal: Nature Communications

Article Title: mTORC1 upregulates B7-H3/CD276 to inhibit antitumor T cells and drive tumor immune evasion

doi: 10.1038/s41467-023-36881-7

Figure Lengend Snippet: a IFN-γ is one of the top activated pathways in B7-H3 knockdown tumors versus control tumors ( n = 3/group). NES = normalized enrichment score. b Heatmap of IFN-γ-induced gene expression in bulk tumors and sorted tumor cells of the indicated groups ( n = 3/group). c Heatmap of unsupervised clustering of MHC-I/II gene expression in sorted tumor cells from each group ( n = 3/group). d Immunoblot analysis of tumor lysates derived from the indicated tumors. ( n = 3/group). e Representative images of E-cadherin (E-cad) and STAT1 immunofluorescent staining of tumor sections ( n = 3/group). Scale bar = 100 μm. f Representative images of E-cad and MHC-II immunofluorescent staining on tumor sections ( n = 3/group). White arrows indicate co-expression in the same cells. Scale bar = 100 μm. g Immunoblot analysis of Tsc2−/− 105K cells stably expressing sh-B7-H3 (1) or sh-B7-H3 (2) and control (Ctrl) or CIITA sgRNAs ( n = 3). h CIITA knockout increases the subcutaneous growth of tumor cells in ( g ) in mice. n = 10 mice/group, means ± SEM, two-way ANOVA with Holm-Sidak’s multiple comparisons test, ** p < 0.01, *** p < 0.001, **** p < 0.0001. i Growth of subcutaneous sh-NC, sh-B7-H3 (1), or sh-B7-H3 (2) Tsc2−/− 105K tumors in strain-matched WT or IFN-γ KO mice. n = 10 mice/group, means ± SD, Mann-Whitney U Test, **** p < 0.0001. Percentage of IFN-γ/TNF-α positive tumor-infiltrating CD8 + ( j ) and CD4 + ( k ) T cells in the indicated tumors. n = 6, 7 or 8/group. Exact n is indicated in Source data file. Means ± SD, one-way ANOVA with Dunnett’s multiple comparisons test, ** p < 0.01, *** p < 0.001. Percentage of IFN-γ/TNF-α in WT splenic CD8 + ( l ) and CD4 + ( m ) T cells after co-culture with Tsc2−/− control, B7-H3 (1) or B7-H3 (2) shRNA 105K cells. n = 3, means ± SD, one-way ANOVA with Dunnett’s multiple comparisons test, ** p < 0.01, *** p < 0.001. n Immunoblot analysis of Tsc2−/− 105K cells stably expressing B7-H3 (1) or B7-H3 (2) shRNA and control (Ctrl) or IFNGR1 sgRNAs ( n = 3). o IFNGR1 knockout increases the subcutaneous growth of tumor cells in ( n ) in mice. n = 10 mice/group, means ± SEM, two-way ANOVA with Holm-Sidak’s multiple comparisons test, * p < 0.05, ** p < 0.01. Source data and exact p values are provided in the Source data file.

Article Snippet: The following primary antibodies were used for immunoblotting at 1:1000 dilution unless otherwise indicated: Phospho-p70 S6 Kinase T389 (CST, Cat#9234), p70 S6 Kinase (CST, Cat#2708), TSC2 (CST, Cat#4308), Phospho-S6 S235/236 (CST, Cat# 2211), S6 (CST, Cat#2217), mTOR (CST, Cat# 2983), Raptor (CST, Cat#2280), Rictor (CST, Cat#2114), Phospho-Akt S473 (CST, Cat#4060), Phospho-Akt T308 (CST, Cat#4056), Akt (CST, Cat# 4685), GAPDH (CST, Cat# 2118), CREB (CST, Cat#4820), STAT1 (CST, Cat#14994), p-STAT1 (Tyr 701) (CST, Cat#9167), human B7-H3 (CST, Cat# 14058), YY1 (CST, Cat#46395), HA-tag (CST, Cat#3724), Phospho-substrate (RXXS*/T*) (CST, Cat# 9614), MHC-II (1:500, LSBio Cat#LS-C204829), β-actin (Sigma, Cat#A1978), YY2 (A-5) (1:500, Santa Cruz, Cat#sc-377008), YY2 (C-10) (1:500, Santa Cruz, Cat#sc-374455), mouse B7-H3 (1:500, R&D Systems, Cat#AF1397), and GFP (Abcam, Cat#ab6556).

Techniques: Knockdown, Control, Gene Expression, Western Blot, Derivative Assay, Staining, Expressing, Stable Transfection, Knock-Out, MANN-WHITNEY, Co-Culture Assay, shRNA

Journal: Science Advances

Article Title: STAT3 haploinsufficiency is associated with autosomal dominant hyper-IgE syndrome

doi: 10.1126/sciadv.adw2464

Figure Lengend Snippet: ( A ) Naive CD4 + cells from Stat3 +/+ and Stat3 +/− mice were cultured in vitro with T H 17 polarizing conditions and analyzed by flow cytometry on day 4 for the indicated cytokine expression. Representative dot plots show intracellular IFN-γ and IL-17A expression after restimulation of cells with PMA and ionomycin. Numbers in dot plots represent cell frequencies. ( B ) Mean (±SD) frequencies of the cells gated in (A) are shown. CD4 + splenocytes from Stat3 +/+ and Stat3 +/− mice were stimulated for different time points with IL-6 (50 ng/ml) and the phosphorylation of STAT3 (p-STAT3) ( C ) and STAT1 (p-STAT1) ( D ) were evaluated by flow cytometry. Mean (±SD) of mean fluorescent intensity (MFI) is plotted. Data are representative of five independent experiments (A and B) or at least three independent experiments (C and D). P values by two-way ANOVA followed by Bonferroni’s test.

Article Snippet: The samples were then blotted with the following antibodies to p-STAT3 (Tyr 705 ; mouse monoclonal), p-STAT1 (Tyr 701 ; rabbit monoclonal), STAT3 (mouse monoclonal), STAT1 (rabbit monoclonal) (all from Cell Signaling Technology), and α-tubulin (mouse monoclonal) (Proteintech).

Techniques: Cell Culture, In Vitro, Flow Cytometry, Expressing, Phospho-proteomics

Journal: Virology journal

Article Title: RUNX1 inhibits the antiviral immune response against influenza A virus through attenuating type I interferon signaling.

doi: 10.1186/s12985-022-01764-8

Figure Lengend Snippet: Fig. 5 RUNX1 attenuated IRF3 and STAT1 signaling. a The mRNA level of TRAF3, RIG-I, MAVS, TBK1, IRF3, and STAT1 in the shRUNX1 and shControl cells were assessed by qRT-PCR. b The mRNA of these genes in A549 cells that were transfected with empty plasmid pCMV-GFP or pCMV-RUNX1 was assessed by qRT-PCR. c, d The protein and the phosphorylation level of IRF3 and STAT1 of these cells were assessed by Western blot. e, f These cells were infected with PR8 (MOI = 5) and collected at 3, 6, and 9 h.p.i. The protein and the phosphorylation levels of IRF3 and STAT1 in these cells were assessed by Western blot. Data are mean ± SD of three independent experiments. Significance is by unpaired T-test; *p < 0.05; **p < 0.01

Article Snippet: Mouse anti-RUNX1(A-2) (sc-365644) antibodies were purchased from Santa Cruz, Dallas, TX, USA; rabbit antiIRF3 (11312-AP) and rabbit anti-STAT1 (10144–2-AP) were purchased from Proteintech, Rosemont, IL, USA; rabbit anti-P-IRF3 (Ser396) (4D4G) antibodies and rabbit anti-P-STAT1 (Tyr701) (D4A7) antibodies were purchased from Cell Signaling, Danvers, MA, USA; mouse anti-GAPDH antibody (AF0006) was purchased from Beyotime, Shanghai, China; mouse mAbs to viral proteins NP and M1 of IAV were obtained from Dr. Jiyong Zhou [35].

Techniques: Quantitative RT-PCR, Transfection, Plasmid Preparation, Phospho-proteomics, Western Blot, Infection