Fig. 2. Clik1 associates with CLP-36. (A) A yeast two-hybrid screen from a human fetal liver cDNA library with LexA-Clik1 identified 37 clones of which 36 represented CLP-36. Three representative clones (38, 51 and 47) with their PDZ (grey) and LIM (black) domains are shown. (B) To demonstrate the association of CLP-36 with cellular Clik1, 300 micrograms of U2OS cell extracts overexpressing Myc-Clik1 or vector control were incubated with GST-CLP-36 or GST proteins. Subsequently proteins associated with the GST proteins were purified and analyzed by anti-Myc western blotting (anti-Myc), demonstrating that CLP-36 associated with Myc-Clik1. Ponceau staining (lower panel) was used to control levels of GST-CLP-36 and GST proteins. The faster migrating bands in Ponceau staining in GST-CLP-36 lanes are caused by degradation of the purified protein. The input panel demonstrates amounts of Clik1 in 50 µg of extracts subsequently used for the pull-down assay.