Fig. 2

Zebrafish parkin promotes auto-/transubiquitylation and is prone to misfolding in the presence of high level oxidative stress.

(A) Zebrafish parkin shows auto-/transubiquitylation activity. HEK293T cells were co-transfected with HA-ubiquitin and either FLAG-tagged human parkin (hu-parkin) or FLAG-tagged zebrafish parkin (zf-parkin). The cells were treated with the proteasomal inhibitor MG-132 overnight. At 24 h after transfection, cells were harvested, lysed under denaturing conditions, and cleared by centrifugation. The supernatants were subjected to an immunoprecipitation (IP) using anti-FLAG agarose beads. The immunoprecipitates were resolved by SDS-PAGE and immunoblotted with an anti-HA mAB (WB). Aliquots of the supernatants were removed before immunoprecipitation and were immunoblotted with anti-FLAG mAB (input, lower panel). (B) SH-SY5Y cells were transiently transfected with FLAG-tagged zebrafish parkin (FLAG-zf-parkin) and exposed to oxidative stress (10 and 20 mM H₂O₂, 30 min). The cells were then lysed in detergent buffer (0.1% Triton X-100 in PBS), and fractionated into detergent-soluble (supernatant) and -insoluble (pellet) fractions by centrifugation. Parkin present in the supernatant and pellet fraction was analyzed by Western blotting using an anti-FLAG monoclonal antibody. GAPDH (only present in the soluble fraction) was used as a loading control. (C) SH-SY5Y cells were transiently transfected with FLAG-tagged zebrafish parkin (FLAG-zf-parkin) and exposed to dopamine stress (0.5, 0.75, 1 and 2 mM, 5 h). The cells were then lysed in detergent buffer (0.1% Triton X-100 in PBS), and detergent-soluble and -insoluble fractions were analyzed as described in (B). Actin was used as a loading control.