Using the STAT1 assay kit or Western blot, STAT1 phosphorylation at Tyr701 is detected in interferon-gamma-treated HeLa cells (+), compared with untreated HeLa cells (-).
HeLa cells were seeded at 40K cells/well in a 96 well tissue culture microplate overnight. The next day cells were treated with various concentrations of interferon-gamma for 20 mins. The medium was removed from the wells, and cells were lysed with 120 µl/well of Lysis Mix, with shaking for 10 min. The lysates were transferred to a PhosphoTracer assay plate and assayed for either phospho-STAT1, using the standard protocol. Signal in the wells was determined using a plate reader.