C/EBP gene expression is regulated by AP-2α and AP-2γ. (A) Immunofluorescence with monospecific C/EBPα and C/EBPβ antibodies in E18.5 skin. Dotted lines denote dermoepidermal boundary. Bars, 20 μm. (B) Immunoblots of protein lysates from 1°MK cultured under 50-μM or 1.5-mM Ca²⁺ conditions. The C/EBPα antibody detects both Cα p42 and Cα p30 isoforms; the C/EBPβ antibody detects LAP* and LAP isoforms. β-Actin is used as a loading control. (C) Analyses of the effects of AP-2 ablation on C/EBP gene expression and promoter activity. Real-time PCRs with primers specific for C/EBPα and C/EBPβ were performed on mRNAs from in vivo epidermis and 1°MK. DcKO levels are expressed as changes versus WT and error bars represent standard deviation for three or more independent experiments. (bottom right) Luciferase assay of C/EBPα promoter activity in 1°MK in high Ca²⁺ media. 1.5 kb of C/EBPα promoter plus 5′-UTR sequences were used to drive firefly luciferase expression (pC/EBPα-pGL3; Tang et al., 1997). Fold change represents pC/EBPα-pGL3 firefly luciferase activity divided by basal level of pGL3 firefly luciferase activity, with cytomegalovirus Renilla luciferase activity as the standardized internal control for transfection efficiency. Three independent experiments performed in duplicate are represented. Error bars represent standard deviations. (D) Effects of ectopic expression of AP-2α, C/EBPα, or C/EBPβ in DKO and WT 1°MK. Where indicated (vector), cells were either transduced 36 h with an IRES-GFP empty vector (control) or an IRES-GFP retroviral expression vector encoding the transcription factor indicated. Transduced cells were purified by FACS, mRNAs were isolated, and real-time PCRs were performed with the primers indicated.