Fig. 1. LAP2ß represses transcriptional activity in a dose-dependent manner. (A) LAP2ß represses the activity of E2F transcription factors. The E2F reporter (0.5 µg) and pCMV-ß-gal (0.5 µg) were co-transfected with expression vectors for E2F1 (0.15 µg), E2F4 (0.15 µg), E2F5 (0.15 µg), DP1 (0.15 µg), DP2 (0.15 µg), DP3 (0.3 µg), and LAP2ß (1 µg) into U2OS cells as indicated. (B) LAP2ß represses transcriptional activity of E2F5-DP3, p53, NF-B and the constitutive RSV promoter in a dose-dependent manner, but not that of the constitutive CMV promoter. The specific reporters (0.1 µg) and pCMV-ß-gal (0.5 µg), together with expression vectors for E2F5 (0.15 µg) and DP3 (0.3 µg, i), p53 (0.1 µg, ii), p65 (0.5 µg, iii), constitutive RSV promoter (0.1 µg, iv), constitutive CMV promoter (0.1 µg, v), and increasing doses of LAP2ß (0.1-2.0 µg) were transfected into U2OS cells as indicated. For each factor, its known endogenous inhibitor was assayed [Rb (1.0 µg), MDM2 (0.5 µg) and IB (1 µg)]. (C) LAP2ß represses the activity of p65 upon TNF stimulation. U2OS cells were transfected with increasing doses of LAP2ß (0.1-2.0 µg) and IB (1.0 µg). 12 hours before harvesting, the cells were stimulated with TNF (200 units). For all reporter assays, the values shown represent the averages ± S.D. of duplicate (A) or triplicate (B,C) readings after the luciferase values were normalized to the ß-galactosidase values and compared with the mock control.