Fig. 2. PKC-dependent MAPK kinase phosphorylation. (A) Immunoblots of total brain extract and of cytosol and membrane fractions prepared from control and RD cells treated with TPA for 30 minutes. (B) Immunoblots of total lysate from RD cells transfected with control vector (CMV) and with constitutively active PKC-expressing vector (A25E) using the same antibodies as those described in the legend for Fig. 1 and with specific antibodies that recognize PKC, ß1 and . The filter was normalized with an antibody specific to p54/JNK2. The data shown are representative of four independent experiments. The increases (fold over the CMV-transfected cells) in phosphorylation levels are indicated for each sample. (C) Luciferase assay for detection of activated c-Jun and Elk1 (see Materials and Methods). RD cells cotransfected with activator plasmid GAL4-jun or GAL4-Elk1 and reporter plasmid GAL4-luc together with empty vector (CMV), WT PKC or DN PKC (dominant-negative version, K368) were left untreated or treated with TPA for 24 hours 1 day after transfection.

Return to article