Figure 2. pThoc1 poly-ubiquitination correlates with NEDD4-1 levels in cells.

A) HeLa cells were transfected with the THOC1 and His-Ub expression vectors (all lanes). In addition, cells were transfected with a wild type NEDD4-1 expression vector (NEDD4-1), a catalytic dead NEDD4-1 mutant (NEDD4-1 mut), or were treated with MG132 as indicated. Total cell protein extracts were purified by metal chelate chromatography and purified protein analyzed for the presence of pThoc1 by western blotting (top panel). The middle panel shows relative input levels of pThoc1. The bottom panel shows relative input levels of NEDD4-1. B) HeLa cells engineered to express tetracycline inducible NEDD4-1 targeted shRNA were transfected with Thoc1 and His-UB expression vectors (both lanes), treated with MG132 (both lanes), or treated with tetracycline. Total cell protein extracts were purified and analyzed as in A). C) Wild type or Nedd4-1 knockout murine embryonic fibroblasts were transfected with the Thoc1 and His-Ub expression vectors, and treated with MG132 as indicated. His-Ub containing proteins were purified and analyzed for the presence of pThoc1 as above. Input levels of pThoc1 and NEDD4-1 were determined by western blotting while β-actin serves as a loading control. The NEDD4-1 immunoreactive band detected in NEDD4-1 null cells is a non-specific background protein that becomes detectable upon MG132 treatment.