NEDD4-1 binds and ubiquitinates pThoc1.

<p>A) 293 cell extracts were immunoprecipitated with a pThoc1 specific antibody or non-specific IgG, and the immunoprecipitates analyzed for the presence of NEDD4-1 by western blotting. Input levels of pThoc1 and NEDD4-1 were assayed by western blotting. β-actin serves as a loading control. B) Immunopurified pThoc1 was mixed with the indicated amount of purified recombinant NEDD4-1 in an in vitro reconstituted ubiquitination reaction. pThoc1 poly-ubiquitination was analyzed by western blotting (top panel). Input levels of pThoc1 or NEDD4-1 (bottom panel) were determined by western blotting.