Figure 9

(A) AICAR inhibits LPS- and Aβ-induced activation of NFκB, AP-1 CREB and C/EBP transcription factors. NFkB luciferase and C/EBP luciferase activities in glial cells transfected for NFkB luciferase (i) or C/EBP luciferase following stimulation with LPS and Aβ (1–42) peptide (figure A) were measured as described under legends to figure 1. Experiments were performed in triplicate and data are expressed as mean ± SEM. *P < 0.05 compared to relative control value was considered significant. AICAR inhibited the LPS- or SMase- and Aβ-induced NFκB, C/EBP, CREB and AP-1 binding activity as seen by EMSA (figure B). EMSA was carried out using the nuclear extracts prepared from astrocyte-enriched glial cells after treatment with LPS or SMase and Aβ for 1 h either in the presence or absence of AICAR. In case of EMSA for NFκB, for clarity, the dried gel was exposed for autoradiography either for longer (24 h) or for shorter (6 h) periods. The top shows the picture of the autoradiogram of the shorter exposure time (Sx) and the bottom shows the longer exposure time (Lx). In figure B(i) polyclonal IgGs specific for NFκB subunits -p65, p52, p50, RelB or cRel were used for supershift experiments with nuclear extracts from LPS, Aβ-treated (1 h) glial cells for binding to γ-³²P-labeled NFκB oligomer. Note the supershifted complexes in lanes 2, 4 and 5 (correspond to -p65, p50 and -RelB proteins). In figure (ii) lanes 2–7, polyclonal IgGs specific for C/EBP α, β, p-β, δ and ε were used in supershift experiments with nuclear extracts from LPS, Aβ-treated (1 h) glial cells for binding to γ-³²P-labeled C/EBP oligomer. ε antibodies from two different stocks were tested (in lanes 6 and 7). Note the supershifted complexes in lanes 2, 3 and 5 corresponding to -α, β and -δ subunits of C/EBP.