Fig. 2

Zebrafish cam-kii inhibitors bound to and inhibited cam-kii α activity. A: The GST-pull down assay. The HEK 293 cells were transfected with pFlag-cam-kiiα plasmid. At 24 hr post-transfection, cell lysates were harvested and subjected to GST-pull down assay with GST or various C-terminal deleted forms of cam-kii inhibitors fusion protein beads as indicated in the presence of Ca²⁺/CaM. The pull-down complexes were subjected to Western blot analysis using anti-Flag antibody. Bottom: The equal amount of GST fusion proteins as visualized by Western blot analysis using anti-GST antibody. B: The co-immunoprecipitation assay. The HEK293 cells were co-transfected pFlag vector (left) or pFlag-cam-kiiα (right) combined with pEGFP, pEGFP-cam-kiin1, or pEGFP-cam-kiin2. At 24 hr post-transfection, cell lysates were harvested and subjected to immunoprecipitation with anti-Flag antibody. The immuno-complexes were probed with anti-GFP and anti-Flag antibody. C: The in vitro kinase assay. The HEK 293 cells were transfected with pFlag-cam-kiiα plasmid. At 24 hr post-transfection, cell lysates were immunoprecipitated with anti-Flag antibody. The immuno-complexes were subjected into kinase assay in the presence of Ca²⁺/CaM and GST, GST-cam-kiin1, or GST-cam-kiin2 fusion protein using myelin basic protein as substrate. These proteins were separated by electrophoresis and the phosphorylation was detected by autoradiography. Flag-cam-kiia denotes Flag-cam-kiiα. The data represent one of at least three independent experiments.