Fig. 3.

Parkin ubiquitinates STEP₆₁ in vivo and in vitro. (A) HEK cells were transfected with STEP₆₁, along with HA-ubiquitin or WT parkin, or with parkin mutants. Ubiquitinated proteins were enriched using tandem ubiquitin binding entity 2 (TUBE2) affinity pull-down and probed with anti-STEP antibody or anti-ubiquitin antibody. STEP₆₁ ubiquitination was increased by WT parkin but decreased by the parkin mutants R275W or G430D (n = 3). (B) GST-STEP₆₁ was ubiquitinated by recombinant parkin in the presence of the E1 ligase UBE1, the E2 ligase UbcH7, and HA-ubiquitin. STEP was immunoprecipitated and probed with anti-HA antibody, which shows HA immunoreactivity for higher-molecular-weight STEP–ubiquitin conjugates (n = 4).