Fig. 2. Nucleotide dependency of G protein interaction with recombinant CSP and J domain. Immunoblot analysis showing binding of G, G_ß and Hsc70 to J-domain-GST fusion protein immobilized on agarose. Fusion proteins were incubated in the absence (-) or presence of rat hippocampal homogenate and 2 mm ATP, ATPS, ADP, GTP, GTPS or GDP at 37°C for 30 minutes. Lane 2 shows fusion proteins incubated with hippocampal homogenate in the absence of nucleotides. Lane 9 shows 15 µg of hippocampal homogenate loaded directly on the gel. The beads were washed, and bound proteins were eluted in sample buffer, fractionated by SDS-PAGE and subjected to western blot analysis. The nitrocellulose membrane was probed with anti-G polyclonal, anti-G_ß monoclonal and anti-Hsc70 monoclonal antibodies. G_ß does not interact with the J domain in the absence or presence of any nucleotide tested. G binds to the J domain in the presence of ATP. These results are representative of six independent experiments.