Figure 2. Time-dependent effect of IL-6 on IGFBP-4 mRNA and protein in primary cultured hepatocytes. After 48 h of culture, hepatocytes were incubated for 6, 10, or 24 h with IL-6 (10 ng/ml). A, Northern blot: Cells were collected after 6, 10, or 24 h and total RNA was extracted. 20 μg of RNA were electrophoresed in each lane and Northern blot analysis was performed as indicated in Materials and Methods. Upper panel, autoradiography; lower panel: densitometric analysis. Data are expressed as the mean ± sem of three separate experiments. *, P < 0.05; **, P < 0.01 vs. time-matched control. B, Ligand blot, Culture medium was collected at the same time as mRNA (6, 10, or 24 h), concentrated and electrophoresed, and ligand blot analysis was performed as described in Materials and Methods. Upper panel, autoradiography; lower panel, densitometric analysis. Data are expressed as the mean ± sem of two separate experiments. Binding Protein-4 Messenger Ribonucleic Acid and Protein**This work was supported by grants from the Fund for Scientific Medical Research (Belgium), from the Fonds Spéciaux de Recherche (Université Catholique de Louvain, UCL) and from the Danone Institute (Brussels). Endocrinology. 2001;142(1):241-248. doi:10.1210/endo.142.1.7903 Endocrinology | Copyright © 2004 by the Endocrine Society 1