Fig. 1

Binding affinity of AIMP2 and DX2 to PARKIN and PARP-1 A Myc-tagged AIMP2 and DX2 DNA plasmid were co-transfected with Flag-parkin in Hek293 cell line. After 24 h, the cells were harvested and cell lysates were immunoprecipitated by flag-antibody. B Flag-tagged AIMP2 and DX2 were transfected with empty vector (EV) or myc-tagged parkin. The expression level of AIMP2 and DX2 were decreased after parkin tansfection at a similar level. C A graph quantifying the ubiquitination of AIMP2 and DX2 by PARKIN. (ns, non- significant) qPCR was performed in triplicate. D AIMP2 and DX2 protein stability were assessed with cycloheximide-treated pulse chase assay. The samples were harvested in a time-dependent manner at 0–4 h and examined using immunoblot assays. The quantification was measured by image J (lower bar graph). E Myc-tagged AIMP2 and DX2 transfected samples were collected separately from cytosol and nucleus. HSP70 and YY1 protein were used as fractionation markers. F AIMP2 and DX2 expression was induced by transfection of each plasmid in SH-SY5Y cells followed by analyses with PARP-1 pull-down assays; DX2 had much higher binding affinity with PARP-1 than AIMP2. (WCE; whole cell extract) G Schematic Fig. about the fate of AIMP2 and DX2. Both AIMP2 and DX2 are substrates for Parkin in the cytosol, and DX2 translocates to the nucleus more easily than AIMP2. (ns, non-significant)