Anti py stat5 antibody

Cells were stained with fluorescently tagged antibodies purchased from eBioscience, BD Biosciences, Tonbo Bioscience, or BioLegend (Supplementary Table 2) and analyzed using a BD LSR II flow cytometer. Flow cytometry data were analyzed using FlowJo software (TreeStar). For intracellular cytokine staining, cells were stimulated for 5 hr with CD3 and CD28 antibodies (5 μg/ml each) in the presence of brefeldin A or monensin, harvested and stained with eBioscience Fixation Permeabilization kit. For intracellular phosphorylated STAT5 staining, cells were stimulated with or without rmIL-2 for 20 min, fixed and permeabilized with 4% PFA followed by 90% methanol, and stained with anti-pY-STAT5 antibody (BD Biosciences). Cell sorting of Foxp3⁺ and Foxp3⁻ cells was performed based on YFP or GFP expression using a BD FACSAria II cell sorter.