Figure 5.

C/EBPβ expression levels in LSMCs increased after treatment with Bt₂cAMP. Primary cultured LSMCs were pretreated with PGE₂ or Bt₂cAMP for 12, 24, and 48 h, and total RNA and nuclear protein were extracted. A, C/EBPβ mRNA expression levels were detected using real-time RT-PCR and standardized to GAPDH mRNA. The average + sem of data from triplicate experiments is reported. *, P < 0.05 (ANOVA). B, Changes in nuclear C/EBPβ protein levels upon treatment of LSMCs with PGE₂ or Bt₂cAMP were detected by immunoblotting. Using an antibody (C-19) that recognizes three C/EBPβ isoforms (C/EBPβ-1, 45 kDa; C/EBPβ-2, 42 kDa; and C/EBPβ-3, 20 kDa), cAMP was shown to induce all of these isoforms. C, Immunoblotting was performed with more specific anti-C/EBPβ antibodies, as indicated. A C/EBPβ-LAP antibody could be used to detect C/EBPβ-2 (42 kDa) more distinctly, whereas phosphorylation of C/EBPβ-2 could be determined using an antibody against C/EBPβ-2 phosphorylated at Thr²³⁵ [P-C/EBPβ (Thr²³⁵)]. Equal loading was confirmed by immunoblotting with an anti-actin antibody.