FIG. 3.

Specific coactivation of v-Rel- and AP-1-mediated transcription by CAPERα. (A) CAPERα coactivates AP-1-mediated transactivation. Luciferase assays were performed in 293T cells cotransfected with vectors encoding AP-1 complex components c-Jun and c-Fos (0.2 μg each) along with CAPERα-Flag (4.0 μg), a 4× AP-1-luciferase reporter (1.6 μg), and the hRL-null Renilla internal control (0.01 μg). The total amount of transfected DNA was kept constant (6.01 μg) by addition of pCMV (for transcription factors) or pcDNA3.1(+) DNA (for coactivators). Luciferase activity was normalized to total protein concentration and internal Renilla luciferase activity and is represented as relative light units (RLU). The averages from three independent assays are shown with standard deviations. (B) CAPERα strongly synergizes v-Rel-mediated transactivation. Assays were performed as for panel A in cells cotransfected with v-Rel (0.4 μg) and/or CAPERα-Flag DNA (4.0 μg), along with an IL-6κB-luciferase reporter (1.6 μg) and hRL-null Renilla internal control (0.01 μg). The data represent the averages from three independent assays. (C) Western blot showing that transfection of increasing amounts of CAPERα-Flag does not alter v-Rel expression levels, as seen by probing with an antibody to the RHD. (D) Coactivation of IL-6-κB-luciferase expression by CAPERα is Rel/NF-κB dependent. Luciferase assays were carried out as for panel A in cells cotransfected with v-Rel (0.4 μg) and/or CAPERα (1.2 μg) expression vectors in the presence or absence of IκBαM (3.6 μg), together with IL-6κB-luciferase (0.8 μg) and the hRL-null Renilla control (0.01 μg). (E) CAPERα fails to synergize E2F1-mediated transactivation. Luciferase assays were performed in cells transfected with an expression vector for transcription factor E2F1 (0.4 μg), CAPERα (4.0 μg), or both, along with a p73-luciferase reporter (1.6 μg) and hRL-null Renilla internal control (0.01 μg). The averages from three independent experiments are shown.