特異性：HeLa cells were seeded at 40,000 cells/well and cultured overnight at 37 degree C, 5% CO2, in DMEM with 10% FBS. Cells were treated with either IFN-alpha for 30 min (stimulation) or with JAK inhibitor 1for 30 min then 1.5 nM IFN-alpha (inhibition). The media was removed from the wells, and cells were lysed with 50 uL/well of 1X Lysis Buffer #2 containing 1 mM NaF and 2 mM Na3VO4, on an orbital shaker (400 rpm) for 30 min at room temperature. The lysates were then analyzed for total-STAT3 using the two-plate assay protocol. Lysates analyzed on separate wells for phospho-STAT3 (Y705) are also shown for comparison. The TR-FRET signal was recorded at 665 and 615 nm (EnVision lamp excitation) after a 4 h incubation period at room temperature.
