Fig. 4. Molecular characterization of PDZRN3. (A) Lysates of C2C12 myotubes as well as of HeLa cells infected with an adenovirus containing mouse PDZRN3 cDNA were subjected to immunoblot analysis with polyclonal antibodies to PDZRN3. The positions of molecular size standards are indicated on the left. (B) Lysates of C2C12 myotubes were subjected to immunoprecipitation (IP) with rabbit anti-Semaphorin 4C or control immunoglobulin G (IgG) and the resulting precipitates were subjected to immunoblot analysis with monoclonal antibodies to PDZRN3 or to Semaphorin 4C. Myotube lysates were also subjected directly to immunoblot analysis (Input). (C) GST or GST fusion proteins containing the RING-finger domain of PDZRN3 (GST-PDZRN3-N) or of ARD1 (GST-ARD1-N) were incubated with E1 or E2, as indicated, in a ubiquitination assay. The reaction mixtures were then subjected to immunoblot analysis with anti-ubiquitin (right panel). The purity of the GST and GST fusion proteins used for the ubiquitination assay was evaluated by SDS-PAGE and staining with Coomassie brilliant blue (CBB, left panel).