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Fig. 5

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Immunoprecipitation of MAPK activity in cultured rat hepatic stellate cells exposed to MDA and/or acetaldehyde. One microgram of polyclonal antibodies to ERK, JNK, or p38 were added to cell lysate bound to protein A-agarose beads. The kinase assay was initiated by addition of 1 μl myelin basic protein (MBP) for ERK activity and 5 μl of ATF-2 for either JNK or p38 activity. These studies were performed three times in triplicate for each MAPK activity. Equal protein loading was based on Lowry protein assay [25]. Assays were subjected to 10% SDS-PAGE and autoradiography [26]. Lane 1: Cell exposure to both 200 μM MDA and 200 μM acetaldehyde (AC). Lane 2: Exposure to 200 μM MDA alone. Lane 3: Exposure to 200 μM AC alone. Lane 4: SF media only, control (C). JNK activity was significantly higher in the treatment groups (lanes 1–3) compared to control (lane 4). P38 activity was not significantly different between treatment groups (lanes 1–3) compared to control activity. ERK phosphorylation activity, as measured here by MBP phosphorylation, was barely detectable. Phosphorylation activity between treatment groups was not significantly different.

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