FIG. 4.

In vitro assays. (A) Luciferase assay. NIH 3T3 cells (5 × 10⁵ cells in a 35-mm-diameter dish) were transiently transfected (as described in the legend to Fig. 3A) with RcCMV, Stat3, Stat3Y, Stat3DB, Stat3S, v-src plus RcCMV, v-src plus Stat3, v-src plus Stat3Y, v-src plus Stat3DB, or v-src plus Stat3S. All were transfected with 1 μg of Ly6E (three copies) Luc (34) and a β-galactosidase expression vector. One microgram of RcCMV-based vectors was used per transfection; 500 ng of pBabe/v-src was used in appropriate transfections. Twenty hours after transfection, the cells were fed with DMEM plus 10% BCS. Twenty-four hours later, the cells were lysed and luciferase assays were performed. Each bar represents the average of eight individual transfections with standard deviations, each performed in duplicate and normalized to β-galactosidase activity. (B) FLAG Western blotting. Thirty micrograms of protein per lane from whole-cell extracts from transiently transfected NIH 3T3 cells or v-src-transformed cells (as in Fig. 5A) were analyzed by Western blot analyses using chemiluminescence. The Stat3DB-FLAG construct is slightly smaller (see Fig. 1C) and therefore migrates faster. Stat3S expression was not determined due to its lack of a FLAG epitope. (C) Stat3 Western blotting. As in panel B, 30 μg of protein per lane was analyzed for the relative levels of Stat3 protein. Lanes 1 and 6 reveal endogenous levels of Stat3 protein, while the other lanes demonstrate that the transiently transfected cells are producing additional Stat3 protein. (D) Gel shift. NIH 3T3 cells were transiently transfected as described in the legend to Fig. 5A. Nuclear extracts from these cells were used in an EMSA using a ³²P-labeled Stat3 binding site (M67) (31). Stat3-Stat3 homodimers form when Stat3 is phosphorylated on tyrosine 705. M2 (FLAG) monoclonal antiserum was used at a 1:100 dilution for supershifting of Stat3-FLAG complexes. A 1:100 dilution of Stat3-C antiserum was used for supershifting of Stat3S complexes.