Fig. 3.

Omi decreases the protein levels of L166P, but not wild-type DJ-1 through direct interaction. A Omi interacts with L166P in vitro. Pull-down assays used purified GST, GST-DJ-1, or GST-L166P along with His-Omi expressed in E. coli. Immunoblot analysis used anti-His antibody. B HEK 293 cells co-transfected with HA, DJ-1-HA, or L166P-HA along with V5-S276C were lysed and immunoprecipitated with anti-Omi antibodies. C Omi cleaves L166P in vitro. Cleavage assays were performed using equal amounts of purified GST, GST-DJ-1, or GST-L166P and increasing amounts of purified His-Omi or His-S276C. The proteins were incubated in the protease buffer at 37 °C for 4 h. Immunoblot analysis was performed using anti-GST antibody. D H1299 cells were co-transfected with HA or HA-Omi along with serine-to-alanine mutants of L166P. After 36 h, the cell lysates were subjected to immunoblot analysis with anti-Flag antibody. E Ratios of serine-to-alanine mutants of L166P relative to α-tubulin from (E) from densitometric analysis