Figure 2
Co-expressed endogenous STAT1 is protected from inactivation through heterodimer formation with STAT1-E411A. (A) Kinetics of STAT1 dephosphorylation in HeLa cells expressing green fluorescent protein-tagged wild-type or mutant STAT1. Cells were stimulated with 5 ng/ml IFNγ and subsequently exposed to 500 nM staurosporine for the indicated times. The phosphorylation level was assessed by means of a Western blot using a phospho-tyrosine-specific STAT1 antibody (top panel). The membrane was then stripped and re-incubated with a pan-STAT1 antibody (bottom panel). (B) The E411 mutant resists the inhibitory effects of staurosporine and reacts with co-expressed native STAT1 via heterodimerization. HeLa cells expressing either STAT1-WT-GFP or E411A-GFP were stimulated for 45 min with IFNγ before staurosporine was added for the durations indicated. Whole cell lysates were prepared and incubated in vitro with [32P]-labeled M67 DNA containing a high-affinity STAT binding site. Resulting STAT1-DNA complexes were separated using an electrophoretic gel shift assay. Note the formation of STAT1-GFP homodimers (top arrow), heterodimers of GFP-tagged and native STAT1 (middle arrow), as well as homodimers of native STAT1 (bottom arrow).