FIGURE 5.

Modulatory activity of NELF subunits. A, all NELF subunits reduce the EC₅₀ of gene induction by GR. Representative dose-response curves for Dex induction of GREtkLUC by each NELF subunit in transiently transfected U2OS cells are shown. B, the average -fold changes in GR induction parameters from 4 independent experiments (± S.E.) in U2OS cells treated as in panel B with vector alone (blank), GR and vector (GR), or GR and one NELF subunit. *, p < 0.05, **, p < 0.005, ***, p < 0.0005 versus GR alone. DM, Dex-Mes. C, overexpression of one NELF subunit does not affect the level of the other subunits. Western blots of NELF subunits in U2OS cytosols after transient transfection of each NELF subunit as described under “Experimental Procedures” are shown. FLAG/NELF-E was detected with anti-FLAG antibody. D, levels of GR, TIF2, NELF-B, and NELF-A protein are not affected during overexpression of various factors. U2OS cells were transiently transfected with different combinations of factors under the conditions of the competition assays of Fig. 6. Cytosolic extracts were then analyzed by Western blotting to show that only the level of the transfected protein changes, with β-actin being used as an internal control.