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STAT5 Phosflow

GM Gerdien Mijnheer
LL Lisanne Lutter
MM Michal Mokry
MW Marlot van der Wal
RS Rianne Scholman
VF Veerle Fleskens
AP Aridaman Pandit
WT Weiyang Tao
MW Mark Wekking
SV Stephin Vervoort
CR Ceri Roberts
AP Alessandra Petrelli
JP Janneke G. C. Peeters
MK Marthe Knijff
SR Sytze de Roock
SV Sebastiaan Vastert
LT Leonie S. Taams
JL Jorg van Loosdregt
FW Femke van Wijk
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PBMC and SFMC were thawed and resuspended in PBS (0.5–1.0 × 106 living cells/tube). Surface staining of CD3-BV510 (clone OKT3, 1:400, Biolegend) and CD4-FITC (clone RPA-T4, 1:200, eBioscience) was performed for 25 min at 4 °C. Cells were then stimulated with 0, 1, 10 or 100 IU/ml human (h)IL-2 (Proleukin; Novartis) for 30 min at 37 °C, fixated and permeabilized by using buffers from the Transcription Factor Phospho Buffer Set (BD Biosciences). Intranuclear staining of FOXP3-eF450 (clone PCH101, 1:50), T-bet-eF660 (clone eBio4B10, 1:50; eBioscience), CD25-PE/CY7 (clone M-A251, 1:25) and pSTAT5-PE (clone pY695, 1:25; BD) was performed for 50 min at 4 °C. See Supplementary Fig. 1a and c for gating strategy of FOXP3+ Treg cells and non-Treg cells, and Supplementary Fig. 4h for the gating of pSTAT5. Data acquisition and analysis as above.

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