Figure 4

Biochemical evidence for lack of a cyclic nucleotide dependent kinase in Arabidopsis thaliana. (A) Protein kinase assays using Kemptide as a substrate. Assays were conducted on identically prepared extracts of Arabidopsis and rat adipose tissue in the presence or absence (control) of 10 μM cyclic nucleotide as indicated. Scale is offset in order to visualize both sets of results. All assays were performed in duplicate from three separate preparations and error bars indicate standard error for three separate preparations. (B) Western blotting of extracts with PKA catalytic (PKAcs) and regulatory (RII) subunit polyclonal antibodies. The PKAcs antibody was affinity purified according to [82] and used at 0.5 μg/mL while the RII antibody was used as crude serum at 5000X dilution. Lanes are as follows (A), 10 ng of purified bovine PKAcs or RII, (B) 25 μg clarified crude Arabidopsis extract, (C) 25 μg clarified crude rat adipocyte extract. Positions of mammalian PKA and RII are indicated with arrows.