By J. Gauldie (auth.), M. B. Pepys MA, MD, PhD, FRCP, MCRPath (eds.)
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Extra resources for Acute Phase Proteins in the Acute Phase Response
Fusions between the promoter of several liver-specific genes and the coding region of the bacterial chloroamphenicol acetyl transferase (CAT) gene were constructed and transfected into hep3B cells. The ability of each promoter to stimulate transcription was determined by CAT assays with cell extracts prepared 36 hours after gene transfection (Morrone et al. 1988). Each batch of cells transfected with a particular construct was split into three aliquots. One was incubated with normal medium; a second was treated with 10% MoCM; and the third was treated with recombinant IL-6.
1984). Explanted hepatocytes are, however, more difficult to cultivate. Their life-span in culture is limited and experiments with them are more subject to variability caused in part by damage to the cells during their isolation and in part by contamination with other cell types. More recently, Darlington et al. (1986) showed that conditioned medium from monocytes stimulated for 48 hours with bacterial endotoxin (LPS) also determines in the human hepatoma cell line hep3B the whole spectrum of changes of plasma protein production typical of the APR.
Extract Compo DNA L cell + PMA + HeLa +PMA + Fig. 4. Binding of a PMA induced nuclear transcription factor to the 5' region of the SAA gene. Nuclear extracts were isolated from HeLa cells and transfected L-cells after PMA treatment (50 ng ml- 1 for 4 hours. A Gel retardation assays were performed using the 265 bp 5' fragment of SAA g 9, radiolabelled with 32P. Sequence specific binding was demonstrated by competition with 100 ng of a fragment of the HIV-LTR, containing two copies of the sequence GGGACTTTCC, known to bind the transcription factor NFKB.