Figure 3.

Western analysis of the effects of ribozyme on G_i2α and ERK1/2. G_i2α ribozyme (Rz) or non-cleaving G_i2α ribozyme (Rzm) complexed with DOTAP giving final ribozyme concentrations of 2.5 μM, or only DOTAP (Ctr) were added to hepatocyte cultures at 4–5 hours after the time of seeding. A: Expression of G_i2α protein was assessed after 45 h of ribozyme treatment using antibody (from Calbiochem) directed against C-terminal end of G_i1/i2α . B: Expression of G_i2α and G_qα protein levels in the same samples subsequent to 30 h of ribozyme treatment using antibodies (from NEN™ Life Science Products) against C-terminal sequences of G_i1/i2α or G_qα, respectively. The polyclonal antibodies used to assess G_i2α recognize both the α subunit of G_i2 and G_i1. As shown previously hepatocytes do not express G_i1α [19], so the reactivity with these antibodies reflects only the G_i2α levels. C, D: After 45 h of ribozyme treatment cells were stimulated with or without PGF_2α (10 μM) for 5 min before they were harvested. Immunoblot using antibody against dually phosphorylated ERK1/2 (i.e. ERK1/2-P) (C) is depicted. In Fig. D is developed images from the same immunoblot using antibody detecting total amount of ERK1/2 (i.e. both phosphorylated and unphosphorylated forms) (upper panel) and antibody against dually phosphorylated fractions of ERK1/2 (lower panel).