Expression analysis of inducible STAT1 and transcription factor components in double-transgenic ES cells. ES cells were stably transfected with the constructs EF::DBD-TAD and ZFHD1::mSTAT1. WT ES cells and two representative double-transgenic clones (#A7 and #B5) were treated with 100 nM rapalog or left untreated, lysed and analyzed for inducible STAT1 expression. RT-PCR was performed to detect transgenic STAT1 mRNA and normalized to cyclophilin (A). WB was performed to detect STAT1 protein as described in the legend to Fig. 2(B). Expression of the FRB-p65 fusion protein (TAD; ~48 kDa) in ES cell clone #B5 was determined by WB analysis in comparison to the S2RS #8 MEF cell line. Expression of the DBD fusion protein (~50 kDa) is shown by WB on the rapalog-binding domain FKBP12 in ES cell line #B5 and the MEF cell line S2RS #8 with respective WT cells. Endogenous p65/NFκB (65 kDa) is shown as loading control (C).