Figure 3.

MAPK expression and regulation by TGF-β in activated stellate cells in different liver injury models. Stellate cells were isolated from normal rat livers, BDL for 15 days and after four doses of CCl₄ treatment as described in the Materials and Methods. A: Cell lysates (50 μg total protein) were subjected to immunoblotting using a specific phospho-p38 antibody. The membrane was stripped and reprobed with control p38 antibody. Data from different groups of cells were scanned, quantified, normalized, and presented graphically (n = 3, *P < 0.05 compared to quiescent stellate cells). B: Rats subjected to BDL- and CCl₄-induced liver injury were exposed to STR as in the Materials and Methods or culture-activated stellate cells were incubated with STR as in Figure 1. Cellular proteins (200 μg of total protein) were used for immunoprecipitation with a specific threonine-tyrosine phospho-p38 MAP kinase antibody, followed by detection of phosphorylated p38 MAP kinase-specific substrate; ATF-2 by immunoblotting using phospho-ATF-2 antibody. As controls, cell lysates were subjected to immunoblotting using anti-ATF-2 antibody. Specific bands were scanned, quantified, normalized, and expressed graphically (n = 3, ^#P < 0.05 compared to controls without STR). C: Cell lysates (50 μg of total protein) were subjected to immunoblotting using a specific phospho-SAPK/JNK antibody. Membranes were stripped and reprobed with control SAPK/JNK antibody. For calculation of relative phosphorylation, bands corresponding to JNK 1 (46 kDa) were used and data from different group of cells were scanned, quantified, normalized, and presented graphically (n = 3, *P < 0.05 compared to quiescent stellate cells). D: Rats subjected to BDL- and CCl₄-induced liver injury were exposed to STR as in the Materials and Methods or culture-activated stellate cells were incubated with STR as in Figure 1. An N-terminal C-JUN (1 to 89) fusion protein bound to glutathione Sepharose beads was used to selectively pull down SAPK/JNK from the cellular protein lysate (200 μg of total protein), followed by a kinase reaction. C-JUN phosphorylation was then measured by immunoblotting. As control, cellular proteins were immunoblotted with control C-JUN antibody. For relative quantification both bands for C-JUN phosphorylation (33 and 35 kDa) were used. E: Cell lysates (50 μg of total protein) were subjected to immunoblotting using a specific phospho-ERK antibody. Membranes were stripped and reprobed with control ERK antibody. For relative quantification both specific bands corresponding to phospho-ERK were used and data from different groups of cells were scanned, quantified, and presented graphically (n = 3, *P < 0.05 compared to quiescent stellate cells). F: Rats subjected to BDL- and CCl₄-induced liver injury were exposed to STR as in the Materials and Methods or culture-activated stellate cells were incubated with STR as in Figure 1. Cellular proteins (200 μg of total protein) were used for immunoprecipitation with a specific monoclonal phospho-p44/42 antibody, followed by detection of phosphorylated p44/42 MAP kinase-specific substrate; ELK-1 by immunoblotting using phospho-ELK-1 antibody. As controls, cell lysates were subjected to immunoblotting using anti-ELK-1 antibody. Specific bands were scanned, quantified, normalized to the signal for β-actin, and presented graphically (n = 3, ^#P < 0.05 compared to controls without STR).