p stat2 y690  (Cell Signaling Technology Inc)

Journal: EMBO Reports

Article Title: Immune–epithelial cell cross‐talk enhances antiviral responsiveness to SARS‐CoV ‐2 in children

doi: 10.15252/embr.202357912

Figure Lengend Snippet: A549 ACE2/TMPRSS2 cells deficient for RIG‐I and MDA5 were reconstituted to express only MDA5 or RIG‐I by lentiviral transduction. Cells were mock‐infected or infected with SARS‐CoV‐2 (upper panel: MOI 0.01, lower panel: MOI 1) for 24 h. IFN‐β ( IFNB1 ) expression was determined by qRT‐PCR (bar graph), and MDA5, RIG‐I, and STAT2 protein levels as well as STAT2 phosphorylation (pSTAT2) were analyzed by immunoblotting (lower panel). qPCR values were normalized to 45S rRNA and expressed as mean ± SD, n ≥ 4 (biological replicates, individual experiments shown as dots). Statistical significance between mock and infected samples was tested by a paired two‐tailed t ‐test. * P < 0.05, ** P < 0.01, no asterisk: not significant. Immunoblot representative of two biological replicates. Nasal swabs were taken from healthy individuals of the indicated age groups ( n = 18 children, n = 23 adults) and analyzed by scRNA‐Seq (Loske et al , ). MDA5 ( IFIH1 ) and RIG‐I ( RIGI ) gene expression were quantified across all epithelial cell types of children and adults and displayed as violin plots (median indicated). Statistical significance between all conditions was tested using a two‐tailed Wilcoxon comparison adjusted with a Bonferroni correction. *** P < 0.001, no asterisk: not significant. Source data are available online for this figure.

Article Snippet: The following commercially available antibodies were used: rabbit monoclonal antibodies anti‐p‐IRF3 (S396) (#4947S, RRID: AB_823547), anti p‐STAT1 (Y701) (#7649S, RRID: AB_10950970), anti‐STAT2 (#72604S, RRID: AB_2799824), anti p‐STAT2 (Y690) (#D3P2P, RRID: AB_2773718) were purchased from Cell Signaling Technology.

Techniques: Transduction, Infection, Expressing, Quantitative RT-PCR, Western Blot, Two Tailed Test, Comparison