Figure 3: DJ-1 preferably binds the 20S proteasome and not the 26S proteasome.

(a) 20S and 26S proteasomes purified from rat livers were incubated overnight at 4 °C in the presence (+) or absence (−) of purified recombinant DJ-1. Samples were then separated using 4% native-PAGE, and analysed for peptidase activity, or interaction with DJ-1 by western blot (WB) using anti- α4 (a 20S proteasome subunit), RPN2 (a 19S proteasome subunit) and DJ-1 antibodies. This analysis revealed that DJ-1 co-migrated with the 20S proteasomes, but not with the 26S proteasomes. (b) Lysates of cytosolic proteins from HEK293 cells (L) stably expressing the proteasome β4 subunit tagged with C-terminal FLAG, were subjected to IP using anti-DJ-1 and anti-RPN2 antibodies or anti-FLAG affinity gel and analysed by western blot, using antibodies against DJ-1, α1-subunit and RPN2. Unbound proteins (UB) were run in parallel. The reciprocal co-IP experiments using anti-DJ-1 and anti-FLAG antibodies confirmed the interaction between the 20S complex and DJ-1. The low signal of RPN2 in the DJ-1 IP may indicate that DJ-1 interacts with the singly capped 26S proteasome. Notably, the assay was performed under conditions that preserve the integrity of the 26S proteasome (10% glycerol and 1 mM ATP), as can be seen by the RPN2 bands that appeared when anti-FLAG was used for co-IP. All experiments were repeated at least three times.