Western blot showing specificity of antibodies used for immunofluorescence. A. Gravin was detected using affinity-purified antibodies raised against residues 1130–1780 of the recombinant protein. HeLa cell lysates were incubated with 15 μg of anti-gravin (Lysate) or 15 μg of preimmune IgG (Pre). B. and C. Triton X-100 soluble supernatant (Sup) and insoluble pellet (Pel) of rat intercostal muscle homogenate were subjected to polyacrylamide gel electrophoresis, transferred to membrane, and immunoblotted to detect the protein of interest. Molecular weight markers are shown with arrowheads. Of particular importance are the blots showing that PKC α the major PKC isoform present in muscle, is not detected with the PKC βII antibody, and that there is little overlap between the specificities of the PKA regulatory domain antibodies.