The transcription and translation of wild-type and mutant pCMV-C/EBPβ expression vectors in COS-1 cells. Western blot analysis of the C/EBPβ isoforms synthesized in COS-1 cells transfected with wild-type pCMV-C/EBPβ and pCMV-C/EBPβ mutated at the 20-kDa AUG start site. The AUG was mutated to TTG. The expression vectors contain a Flag tag at their C-terminal ends. Western blot analyses were performed on COS-1 nuclear extracts using anti-Flag (lanes 1–4), anti-C-terminal C/EBPβ-antibodies (lanes 5–8); and anti-N-terminal C/EBPβ antibody (lanes 9–13). Southwestern blot analysis was performed on the COS-1 nuclear extracts used for the immunoblot analyses (lanes 13–16). Lanes 1, 5, 9, and 13, control COS-1 nuclear extract; lanes 2, 6, 10, and 14, nuclear extracts of COS-1 cells transfected with expression vector lacking C/EBPβ sequences (pCMV-B); lanes 3, 7, 11, and 15, nuclear extracts of COS-1 cells transfected with wild-type pCMV-C/EBPβ; lanes 4, 8, 12, and 16, nuclear extracts from COS-1 cells transfected with pCMV-C/EBPβ mutated at 20-kDa AUG start site. Maps of the C/EBPβ expression vectors used in these experiments are shown below the autoradiogram.