(A) Analysis of ectopically expressed BRDT in selected 293T clones. Approximately 20 μg of whole extract isolated from either 293T, 293T/pBABE, or 293T/BRDT-FLAG cells was analyzed by immunoblotting using either anti-FLAG, BRDT, or GAPDH rabbit polyclonal antibodies. (B) Nuclear extracts from 293T/pBABE and 293T/BRDT-FLAG cells were FLAG-immunoprecipitated, and after extensive washing the retained proteins were analyzed by immunoblotting using anti-BRDT and anti-FLAG antibodies. (C) Immunoblot analysis of BRDT-FLAG nuclear and cytoplasmic expression in one of the stable 293T/BRDT-FLAG cell lines. Detection of tubulin served as a cytoplasmic extract control.