total stat1 cell signalling cat# 9176s  (Cell Signaling Technology Inc)

Journal: mBio

Article Title: Combination of proviral and antiviral roles of B cell-intrinsic STAT1 expression defines parameters of chronic gammaherpesvirus infection

doi: 10.1128/mbio.01598-24

Figure Lengend Snippet: Acute MHV68 replication is not affected by B cell-specific STAT1 deficiency. (A) Lysates from splenic B cells sorted from CD19 Cre-negative and -positive STAT1 fl/fl littermates were subjected to western analyses using indicated antibodies. (B, C) CD19 Cre-negative and -positive STAT1 fl/fl littermates were intranasally infected with 10,000 PFU of MHV68. Lungs harvested at 5 ( B ) or 10 ( C ) days post-infection were homogenized and subjected to plaque assay to measure MHV68 titers. Each symbol indicates an individual mouse. Dotted line indicates limit of detection. Here and in subsequent figures, error bars represent the standard error of the mean (SEM).

Article Snippet: The membranes were blocked with 5% non-fat milk (wt/vol) in 1× Tris-buffered saline with 0.05% Tween-20 (TBST) at room temperature for 1 hour and then probed with anti-total STAT1 (1:10,000, Santa Cruz Biotechnology, Dallas, TX) and anti-beta actin (1:10,000, Cell Signaling Technology, Danvers, MA, cat. 4970) antibodies in 1% milk in TBST overnight at 4°C.

Techniques: Western Blot, Infection, Plaque Assay

Journal: mBio

Article Title: Combination of proviral and antiviral roles of B cell-intrinsic STAT1 expression defines parameters of chronic gammaherpesvirus infection

doi: 10.1128/mbio.01598-24

Figure Lengend Snippet: B cell-intrinsic STAT1 expression has anatomic site-specific roles in the regulation of chronic gammaherpesvirus infection. CD19 Cre-negative and -positive STAT1 fl/fl littermates were infected as in and analyzed at 16 days post-infection. (A) Persistent MHV68 replication in lung homogenates. Data were pooled from indicated number of animals in each group. * P < 0.05. Splenocytes ( B, C ) and peritoneal cells ( D, E ) were pooled from three to five animals/group in each study and cell suspensions subjected to limiting dilution assays to define the frequency of MHV68 DNA+ cells ( B, D ) and frequency of ex vivo reactivation ( C, E ). Data were pooled from indicated number ( n ) of independent studies. In the limiting dilution assays, the dotted line is drawn at 63.2%, and the x -coordinate of the intersection of this line with the sigmoid graph represents an inverse of frequency of positive events.

Article Snippet: The membranes were blocked with 5% non-fat milk (wt/vol) in 1× Tris-buffered saline with 0.05% Tween-20 (TBST) at room temperature for 1 hour and then probed with anti-total STAT1 (1:10,000, Santa Cruz Biotechnology, Dallas, TX) and anti-beta actin (1:10,000, Cell Signaling Technology, Danvers, MA, cat. 4970) antibodies in 1% milk in TBST overnight at 4°C.

Techniques: Expressing, Infection, Ex Vivo

Journal: mBio

Article Title: Combination of proviral and antiviral roles of B cell-intrinsic STAT1 expression defines parameters of chronic gammaherpesvirus infection

doi: 10.1128/mbio.01598-24

Figure Lengend Snippet: B cell-intrinsic STAT1 expression supports MHV68-driven germinal center response and selectively promotes generation of MHV68-driven self-reactive antibodies. CD19 Cre-negative and -positive STAT1 fl/fl littermates were infected as in and analyzed at 16 days post-infection. (A, B) Splenic B cells defined as B220 + (representative flow plots in Fig. S3) were measured by flow cytometry with proportion ( A ) and absolute cell number ( B ) shown. (C, D) Splenic germinal center B cells defined as B220 + GL7 + CD95 + were measured with proportion ( C ) and absolute cell number ( D ) shown. (E–H) Sera collected from infected and mock-infected mice of indicated genotypes at 16 days post-infection were subjected to ELISA to measure levels of total IgG ( E ), total IgM ( F ), anti-MHV68 IgG ( G ), and anti-double-stranded DNA IgG ( H ). Each symbol in (A–F) represents an individual animal. In (G and H), “ n ” indicates the number of sera from individual animals that were analyzed with data pooled within each group. * P < 0.05; ** P < 0.01.

Article Snippet: The membranes were blocked with 5% non-fat milk (wt/vol) in 1× Tris-buffered saline with 0.05% Tween-20 (TBST) at room temperature for 1 hour and then probed with anti-total STAT1 (1:10,000, Santa Cruz Biotechnology, Dallas, TX) and anti-beta actin (1:10,000, Cell Signaling Technology, Danvers, MA, cat. 4970) antibodies in 1% milk in TBST overnight at 4°C.

Techniques: Expressing, Infection, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: mBio

Article Title: Combination of proviral and antiviral roles of B cell-intrinsic STAT1 expression defines parameters of chronic gammaherpesvirus infection

doi: 10.1128/mbio.01598-24

Figure Lengend Snippet: B cell-intrinsic STAT1 expression supports MHV68 infection of germinal center B cells. CD19 Cre-negative and -positive STAT1 fl/fl littermates were intranasally infected with 10,000 PFU of MHV68.ORF73βla reporter virus, spleens harvested at 16 days post-infection, and pooled from three animals within each group within each independent experiment. Infected cells were defined as positive for cleaved CCF2 β-lactamase substrate (indicated as CCF2 + ). (A) Representative gating strategy for CCF2 + B220 + splenocytes. (B) Percent and absolute number of infected splenic B220 + cells. (C) Representative gating strategy for germinal center B cells (B220 + GL7 + CD95 + ) subsequently gated for cleaved CCF2 substrate (CCF2 + ) to define infected germinal center B cells. (D) Percent and absolute number of infected germinal center B cells. In (B and D), each symbol represents analysis of splenocytes pooled from three to five mice/genotype in a single experiment; the connecting lines represent paired observations within a single study. Results of three studies are pooled. * P < 0.05; ** P < 0.01.

Article Snippet: The membranes were blocked with 5% non-fat milk (wt/vol) in 1× Tris-buffered saline with 0.05% Tween-20 (TBST) at room temperature for 1 hour and then probed with anti-total STAT1 (1:10,000, Santa Cruz Biotechnology, Dallas, TX) and anti-beta actin (1:10,000, Cell Signaling Technology, Danvers, MA, cat. 4970) antibodies in 1% milk in TBST overnight at 4°C.

Techniques: Expressing, Infection, Virus

Journal: mBio

Article Title: Combination of proviral and antiviral roles of B cell-intrinsic STAT1 expression defines parameters of chronic gammaherpesvirus infection

doi: 10.1128/mbio.01598-24

Figure Lengend Snippet: B cell-intrinsic STAT1 expression attenuates latent infection of peritoneal B cells. CD19 Cre-negative and -positive STAT1 fl/fl littermates were intranasally infected with 10,000 PFU of wild-type MHV68. At 16 days post-infection, peritoneal cells were pooled from three to five mice/group in each experiment, pooled cells sorted into CD19 + B cells and CD19 − non-B cells using magnetic beads, and sorted populations subjected to limiting dilution PCR assays. Data were pooled from two to three independent experiments. The dotted line is drawn at 63.2%, and the x -coordinate of intersection of this line with the sigmoid graph represents an inverse of frequency of positive events.

Article Snippet: The membranes were blocked with 5% non-fat milk (wt/vol) in 1× Tris-buffered saline with 0.05% Tween-20 (TBST) at room temperature for 1 hour and then probed with anti-total STAT1 (1:10,000, Santa Cruz Biotechnology, Dallas, TX) and anti-beta actin (1:10,000, Cell Signaling Technology, Danvers, MA, cat. 4970) antibodies in 1% milk in TBST overnight at 4°C.

Techniques: Expressing, Infection, Magnetic Beads

Journal: mBio

Article Title: Combination of proviral and antiviral roles of B cell-intrinsic STAT1 expression defines parameters of chronic gammaherpesvirus infection

doi: 10.1128/mbio.01598-24

Figure Lengend Snippet: B cell-specific attenuation of type I IFN signaling does not fully phenocopy splenic viral and host phenotypes driven by B cell-specific STAT1 deficiency during chronic MHV68 infection. (A) CD19-Cre IFNAR1 fl/fl mouse model validation. Splenocytes isolated from naïve IFNAR1 fl/fl mice of the indicated CD19 Cre genotypes were sorted into CD19 + B and CD19 − non-B cells using magnetic beads. Sorted populations were incubated for 4 hours in the presence or absence of 10 U/mL of recombinant mouse IFNβ. At the end of incubation, RNA was isolated and subjected to quantitative reverse transcription-PCR (qRT-PCR) to measure mRNA levels of MX-1, with subsequent normalization to corresponding GAPDH mRNA levels. Each symbol represents an individual spleen. ** P < 0.01; * P < 0.05. (B–G) CD19 Cre-negative and -positive IFNAR1 fl/fl littermates were intranasally infected with 10,000 PFU of wild-type MHV68 and analyzed at 16 days post-infection. (B) Persistent MHV68 replication in lung homogenates. Data were pooled from indicated number of animals per group. (C, D) Splenocytes were pooled from three to five animals/group in each experiment and cell suspensions subjected to limiting dilution assays to define the frequency of MHV68 DNA+ cells ( C ) and frequency of ex vivo reactivation ( D ). Data in (C, D) were pooled from the indicated number of independent experiments. In the limiting dilution assays, the dotted line is drawn at 63.2%, and the x -coordinate of intersection of this line with the sigmoid graph represents an inverse of frequency of positive events. (E–G) Splenic germinal center B cells defined as B220 + GL7 + CD95 + [representative flow plot of MHV68-infected mice in (E)] were measured by flow cytometry with proportion ( F ) and absolute cell number ( G ) shown. Each symbol in (F, G) represents an individual animal. ns, not significant.

Article Snippet: The membranes were blocked with 5% non-fat milk (wt/vol) in 1× Tris-buffered saline with 0.05% Tween-20 (TBST) at room temperature for 1 hour and then probed with anti-total STAT1 (1:10,000, Santa Cruz Biotechnology, Dallas, TX) and anti-beta actin (1:10,000, Cell Signaling Technology, Danvers, MA, cat. 4970) antibodies in 1% milk in TBST overnight at 4°C.

Techniques: Infection, Biomarker Discovery, Isolation, Magnetic Beads, Incubation, Recombinant, Reverse Transcription, Quantitative RT-PCR, Ex Vivo, Flow Cytometry

Journal: bioRxiv

Article Title: Selective epigenetic regulation of IFN-γ signature genes by JAK inhibitor in inflammatory diseases

doi: 10.1101/2024.08.05.606293

Figure Lengend Snippet: (A) Flow cytometry assessment of STAT1 and STAT3 protein levels in THP-1 cells treated with PMA and IFN-γ (or without) at various time points. Kinetics of total STAT and phospho-STAT tyrosine (STAT1, Tyr701; STAT3, Tyr705) measured by flow cytometry in IFN-γ-primed macrophages compared to resting macrophages. (B) Kinetics of total STAT1 (or STAT3) and phospho-STAT1 (or STAT3) measured by flow cytometry in JAK inhibitor-treated macrophages compared to resting and JAK inhibitor-untreated macrophages. Resting and IFN-γ-primed macrophages were differentiated for 24 h and then treated with JAKi (or DMSO) for up to 6 h. (A and B) Mean fluorescence intensity (MFI) represents a fold change compared to unstained samples. (C) RT-qPCR analysis of normalized target mRNA relative to TBP mRNA in THP-1 monocyte-derived macrophages under indicated conditions. IFN-γ-primed macrophages were treated with JAK inhibitor at a concentration of 1 μM for up to 6 h. Data show means ± SD from two independent experiments. (D) K-means clustering of differentially expressed (DE) genes in pairwise comparisons between the four conditions. DE genes identified by EdgeR (FDR adjusted P < 0.05, fold change > 2) were used. TPM values of RNA-seq data were filtered to be greater than 4. Non-significant clusters between replications were removed, resulting in three identified clusters. Clusters are indicated on the left. (E-G) Examples of expression for selected genes from clusters identified in the heatmap. Each dot on the bar plot represents one sample, and error bars denote the standard deviation. Error bars represent means ± SD. (H) Gene ontology (GO) analysis of THP-1 RNA-seq using genes positively correlated with HMDM RNA-seq. Heatmap displays the P-value (-Log10) significance of GO term enrichment for genes in each cluster, with clusters shown at the top. Downregulated by IFN-γ, n = 42; JAKi-sensitive, n = 129; JAKi-insensitive, n = 112. (I) Identification of genes associated with JAKi-sensitive or JAKi-insensitive in RA patients. Clusters are indicated on the left. (J) Identification of genes associated with JAKi-sensitive or JAKi-insensitive in COVID-19 patients. Clusters are indicated on the left. For heatmaps of single cells, mean expression values were used, and hierarchical analysis was performed. (K) GO analysis of overlapping JAKi-sensitive and JAKi-insensitive genes in RA or COVID-19 patients. Clusters are indicated on the left. JAKi-sensitive, n = 51; JAKi-insensitive, n = 24. p < 0.05(*), p < 0.01(**), p < 0.001(***) and p < 0.0001(****) by one-way ANOVA. GO analysis was performed using Metascape ( http://metascape.org/ ).

Article Snippet: Add antibody (Alexa Fluor® 647 Mouse anti-Total Stat1, BD biosciences, 58560; PE anti-STAT1 Phospho (Tyr701), BioLegend, 666404; APC Mouse anti-Total Stat3, BD biosciences, 560392; PE/Cyanine5 anti-STAT3 Phospho (Tyr705), BioLegend, 651014) cocktail(s) to appropriate tubes, vortex to mix, and incubate for 30 minutes at room temperature in the dark.

Techniques: Flow Cytometry, Fluorescence, Quantitative RT-PCR, Derivative Assay, Concentration Assay, RNA Sequencing Assay, Expressing, Standard Deviation