anti total stat1  (Santa Cruz Biotechnology)

Journal: The Journal of Biological Chemistry

Article Title: The stress-activated kinase p38 mediates non-canonical activation of Src and tyrosine phosphorylation of the adapter protein TAB1

doi: 10.1016/j.jbc.2026.111200

Figure Lengend Snippet: p38 enhances Src-mediated TAB1-Y481 phosphorylation. A and C , COS-7 cells were transfected with EGFP-TAB1, Src, and the wild-type (WT) or kinase-dead (KD) mutant of Flag-p38α. SB203580 (10 μM) was added for 5 h ( C ). B , The relative quantification of pY481-TAB1, normalized to total TAB1, is presented as the mean ± SD of three independent experiments. D – F , COS-7 cells transfected with EGFP-TAB1 (WT or YF) were stimulated with 1 mM H 2 O 2 for the indicated time. SB203580 was added as a pre-treatment for 5 h. G , COS-7 cells were pretreated with baricitinib (0.5 μM), SB203580 (10 μM), and saracatinib (0.5 μM) for 1 h and then treated with 1 mM H 2 O 2 for 5 min. H , The relative quantification of pY701-STAT1, normalized to total-STAT1, is presented as the mean ± SD of three independent experiments. Cell lysates were immunoblotted with primary antibodies indicated ( A and C – G ). p values were calculated by one-way ANOVA followed by either Tukey’s HSD test was applied. ∗ p < 0.05, ∗∗ p < 0.01.

Article Snippet: Primary antibodies for pY419-Src family kinase (2101S), total-Src (2110S), pT180/pY182-p38 (4511S), pY701-STAT1 (sc-136229), total-STAT1 (sc-346), pY576/577-FAK (3281S), and total-FAK (3285S) were purchased from Cell Signaling Technology. pS75-Src (ab79308) was from Abcam.

Techniques: Phospho-proteomics, Transfection, Mutagenesis, Quantitative Proteomics

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