Anti p stat3 s727 pe

Stimulated T cells and IL-12 receptor-specific siRNA-transfected T cells were stained with anti-mouse CD4-peridinin-chlorophyll protein and anti-mouse CD25-allophycocyanin for 30 min at 4 °C. The cells were permeabilized and fixed with CytoPerm/CytoFix (BD Biosciences, San Diego, CA, USA) in accordance with the manufacturer’s protocol; further stained with anti-mouse Foxp3-phycoerythrin (PE) and/or anti-mouse IL-17-fluorescein isothiocyanate (FITC), anti-mouse IFN-γ-PE, and anti-mouse IL-4-PE (all from eBioscience, San Diego, CA, USA); and subjected to flow cytometric analysis (FACSCalibur; BD Biosciences). For p-STAT3 Y705 and S727 and p-STAT5 analysis, splenocytes were treated with p40-EBI3 (1 and 10 μg/mL) and IL-6 for 1 h; the cells were then stained with anti-mouse CD4-FITC. The cells were fixed and permeabilized with Lyse/Fix Buffer (BD Pharmingen, San Jose, CA, USA) and Perm Buffer II (BD Pharmingen). The permeabilized cells were stained with anti-p-STAT3 Y705-PE, anti-p-STAT3-S727-PE, or anti-p-STAT5-PE (BD Pharmingen) and subjected to flow cytometric analysis (CytoFLEX; Beckman Coulter, Fullerton, CA, USA).

Macrophages were stained after inducing ER stress for IL-10R expression using anti-IL10Rα-PE antibody (CD210, REA239, Miltenyi Biotec) in PBS containing 0.5% BSA. For intracellular staining cells were stimulated with IL-10 or IL-6 for 15 min and subsequently, fixed with 4% paraformaldehyde (Thermo Scientific) for 15 min at 37 °C washed and permeabilized using ice-cold methanol for at least 60 min at − 20 °C [19 (link)]. Staining was done using the following antibodies: anti-pSTAT3(S727)-PE (558557; BD Biosciences), anti-pSTAT3(Y705) (9145S; Cell Signaling), anti-STAT3 (12640S; Cell Signaling), anti-pSTAT3(S727) (94994S; Cell Signaling), and anti-STAT3-FITC (IC1799F; R&D Systems). Fluorescence was measured using a FACSCanto II (BD Biosciences).