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. 2012 Jul 9;7(7):e38837. doi: 10.1371/journal.pone.0038837

Figure 3. Although Parkin can interact with both ARTS and Sept4_i1, it selectively ubiquitinates only ARTS. AI, II.

Figure 3

. Parkin promotes degradation of ARTS through the Ubiquitin Proteasome System (UPS). COS-7 cells co-transfected with 1myc-Parkin and AU5-ARTS were treated with the proteasome inhibitor, MG132. Expression levels of ARTS were significantly decreased in the presence of Parkin, and were restored upon addition of MG132. AII. Densitometry analysis of WB results shown in AI. Protein levels were normalized to actin. Graph presents mean ± SEM of the densitometry values relative to actin levels. B. Parkin serves as an E3-ligase for ARTS. COS-7 cells co-transfected with 1myc-Parkin, AU5-ARTS and HA-8xubiquitin were subjected to in vivo ubiquitination assay followed by immunoprecipitation (IP) with either monoclonal anti-ARTS antibody or anti-Sept4_i1 antibody. Non-transfected cells and cells transfected with only 1myc-Parkin or AU5-ARTS served as controls. ARTS is ubiquitinated in the presence of Parkin. C. Parkin binds both ARTS and Sept4_i1. COS-7 cells were co-transfected with 1myc-Parkin together with Flag-Sept4_i1 or AU5-ARTS. Lysates were subjected to IP with anti-Parkin antibody followed by WB analysis. D. COS-7 cells treated with the proteasome inhibitor, MG132 were co-transfected with 1myc-Parkin or mutant Parkin T240R together with 6myc-ARTS or Flag -Sept4_i1. Immunoprecipitation was done with either anti-ARTS or anti-Sept4_i1 antibody followed by In vivo ubiquitination assay. Western blot analysis was performed with anti-ubiquitin antibody (see methods). Parkin selectively ubiquitinates ARTS but not Sept4_i1. Mutant T240R Parkin lacking E3-ligase activity, demonstrates markedly decreased ability to ubiquitinate ARTS as compared to wt Parkin. E. COS-7 cells were transiently transfected with ARTS, Parkin, and the mutant Parkin T240R with compromised E3-ligase activity. The levels of cleaved caspase-3 (cCasp3) representing rate of apoptosis were visualized using western blot analyses. Co-transfection of COS-7 cells with ARTS and Parkin resulted in ARTS degradation and apoptotic suppression, as visualized by the absence of cleaved caspase-3 (cCasp3). In contrast, co-transfection with ARTS and mutant Parkin T240R restored apoptosis in these cells.