Fig. 2
Increased tyrosine phosphorylation and impaired nuclear accumulation of STAT1-∆N. A Isolated splenocytes from WT mice and tumor cells from STAT1-∆N mice were treated with 50 ng/ml of murine IFNγ and 10 mg/ml of LPS for 30 min. Representative Western blot data from whole protein extracts were stained with p-STAT1, STAT1, p-STAT3, STAT3, NF-κB, and IκBα antibodies, as indicated. B Western blot bands were quantified using ImageJ software from three independent experiments. Asterisks indicate significant differences between the two genotypes depending on the treatment condition. C EMSA with total protein extracts from splenocytes unstimulated or stimulated with LPS and/or IFNγ were incubated with a radioactively [33P]-labeled M67 probe that contains a single consensus GAS motif. The autoradiogram (left) shows a representative result from one of the three independent gel-shift assays used for quantification (right). D Reduced nuclear accumulation of tyrosine-phosphorylated STAT1-∆N following cytokine stimulation. Immunocytochemical experiments were performed using mesenchymal embryonic fibroblasts expressing STAT1-WT or STAT1-∆N; the samples were additionally stained with Hoechst dye for nuclear localization. Fluorescence micrographs show the intracellular distribution of pan-STAT1 in cells expressing the WT protein or its N-terminally truncated variant