(a) Expression of ATF2^50–100 increases expression of c-Jun. Protein extracts prepared from indicated cells were subjected to immunoprecipitation with antibodies to c-Jun followed by immunoblot analysis using antibodies to phosphorylated c-Jun or control non-phosphoantibodies. Parallel analysis was carried out using antibodies to ATF2, c-Fos, and β-actin. Slow-migrating bands in ATF2 Western blots are likely to represent covalently modified forms of ATF2. (b) ATF2-siRNA alters c-Jun and ATF2 expression and the activity of Jun2-luc. SW1 cells were cotransfected with ATF2-siRNA and Jun2-luc as well as β-gal constructs. A portion of the same transfectants was taken for analysis of ATF2 and c-Jun expression to confirm inhibition of ATF2 by siRNA (lower panels). Luciferase assays carried out to monitor changes in TRE-mediated transcription are indicated following their normalization to β-gal. For the three experiments shown, P = 0.0167. (c) ATF2-siRNA alters c-Jun and ATF2 expression and the activity of TRE-luc. Experiment was performed as indicated in panel b, except that TRE-luc was used. For the three experiments shown, P = 0.0027. P-ATF2, phospho-ATF2; P-c-Jun, phospho-c-Jun.