FIG. 5.

Nuclear translocation of SRC-3 is induced by TNF-α. (A) The proteins from untreated cells and cells treated with TNF-α for the times indicated (all grown in DMEM containing 0.5% FBS) were separated into cytoplasmic and nuclear fractions. The membrane containing the fractions was subjected to sequential Western blot analysis by stripping and reprobing the membrane with the indicated antibodies (left panel). The degradation of IκB as evidenced by Western blot analysis was used to monitor the effect of TNF-α on these cells. The results shown are representative of two independent experiments with similar results. (B) Bar chart showing data from experiments as described for panel A. Values corresponding to the y axis represent the percentages of SRC-3 from nuclei (nuclear/nuclear + cytoplasmic [N/N+C]), whereas those corresponding to the x axis represent durations of TNF-α treatment. (C) The proteins from TNF-α-treated HeLa cells were fractionated as described for panel A and assayed by Western blot analysis (left panel). Furthermore, the cytoplasmic fraction was mixed with the nuclear fraction and run side by side with each separated fraction to compare levels of mobility (right panel). (D) Where indicated (+), the nuclear extract was treated with λ-phosphatase (λ-PPase) prior to Western blot analysis. The differences in mobility are indicated by a bracket. N, nucleus; C, cytoplasm.