Figure 6.

Activation of C/EBP transcription complex. (a) Gel shift analysis of C/EBP. Nuclear extracts were prepared from Raw264.7 cells cultured with LPS (1 μg ml⁻¹) in the presence or absence of sauchinone (Sau, 3 μM) for 3 h. All lanes contained 5 μg of nuclear extracts and 5 ng of labeled C/EBP consensus sequence. The specificity of C/EBP binding was confirmed by supershift analysis using the antibodies directed against C/EBP (α, β, δ, ɛ) and p300 proteins. The arrow (left) shows C/EBP complex and SS indicates supershift of the retarded NF-κB band. The specificity of C/EBP binding was confirmed by the addition of an excess amount of free probe (20 ×). (b) The levels of C/EBPβ and δ proteins in the nuclear fraction. Sauchinone (3 μM) inhibited the increase in the level of nuclear C/EBPβ. The level of C/EBPδ in the nuclear fraction was not notably changed. Equal loading of proteins was verified by actin immunoblotting.