Th2 Differentiation and STAT6 Knockdown

Naïve CD4⁺ T cells were isolated and differentiated toward a Th2 phenotype as described above. After 8 days of differentiation, cells were transfected with 100pmol siRNA targeting STAT6 (Invitrogen stealth RNA, forward 5’-CCAAAGCCACUAUCCUGUGGGACAA-3’, reverse 5’-UUGUCCCACAGGAUAGUGGCUUUGG-3’) or control oligonucleotide (AllStars Negative Control siRNA, Qiagen, Hilden, Germany) using an Amaxa Nucleofector Device I and a Human T cell nucleofector kit (Lonza, Szabo Scandic, Vienna, Austria) as described before (25 (link)), and then left incubating for three days in medium containing 100U/ml IL-2 (Immunotools, Friesoythe, Germany). Three days post-transfection, cells were transferred into fresh medium and either restimulated under Th2-conditions or left untreated for 24 hours, before they were lysed in 2× Laemmli SDS sample buffer (Bio-Rad, Vienna, Austria) for Western blot analysis or in TRI Reagent (Sigma, Vienna, Austria) for mRNA extraction and subsequent q-RT-PCR.