Additional file 1: Figure S1. Yeast parkin model. (A) Schematic representation of parkin domains with all cysteine residues indicated. (B) Spotting assays were performed to assess the effects of parkin C-terminal YFP tags compared to wild type parkin in the absence and presence of PINK1. Quantification of three independent experiments is shown in the right part of the panel (p = 0.05). (C) Growth curve of parkin-YFP co-expressed with PINK1 was performed by growing in inducing selective media at 30°C. At mid-log growth all samples were statistically different from the control with a p value ≤ 0.01 as determined from a One-way ANOVA.
Additional file 1: Figure S2. Stress treatment alters parkin localization in yeast and mammalian cells. (A) Spotting assays and fluorescence microscopy of yeast cells expressing C- terminally YFP tagged parkin in the presence of various stress reagents; peroxynitrite , antimycin A, DTT and radiciccol. (B) Apoptosis percentage in SH-SY5Y cells treated with H2O2 and CCCP was determined by the TUNEL assay. Staurosporine as a positive control induced apoptosis in N2a cells and SH-SY5Y cells to 47.7 and 53.4 %, respectively. A one-way ANOVA followed by a Tukey’s multiple comparisons test was used for the analysis with a p value ≤ 0.001. (C) Western blot analysis of protein extracts from yeast expressing parkin grown in the presence of NEM. (D) Western blot analysis of protein extracts from yeast expressing parkin under non- reducing conditions reveals a predominant 50 kDa with a smear signal above and below the predominant band. (E) Western blot analysis of protein lysates from stably transfected FLAG- parkin (HEK 293) in the presence of 100μM H2O2, 10mM AZC and 10 mM MG132 A were probed against parkin under non-reducing and reducing SDS–PAGE conditions. Tubulin was used to assess equal loading for extracts.
Additional file 1: Figure S3. (A) N2a cells transiently transfected with parkin-YFP were analyzed by fluorescence microscopy. (B) Spotting assays were performed with yeast cells expressing C- terminally YFP tagged parkin in a selected set of yeast deletion strains of genes involved in oxidative stress control.
Additional file 1: Figure S4. Parkin aggregation by stress treatment. N2a Cells and SH-SY5Y cells transiently transfected with parkin-YFP treated with the indicated reagents were analyzed by microscopy. Parkin and TOM20 were visualized using an AlexaFluor-488 and 568 conjugated secondary antibody, respectively. The nuclei were stained by DAPI.
Additional file 1: Figure S5. Truncations cause subcellular accumulation of parkin in yeast and mammalian cells. (A, B) Spotting assays and growth curves performed of yeast transformed with C-terminally YFP tagged parkin truncation variants (A) and co expression of C- terminally YFP tagged parkin truncation variants and PINK1 (B). At mid-log growth all samples were statistically different from the control with a p value ≤ 0.01 as determined from a One-way ANOVA. (C) Western blot analysis was performed for detecting parkin. Primary antibodies with epitopes specifically recognizing various domains were used to detect parkin and the various truncation variants and compare relative expression. A RING2/Rcat specific antibody was used to detect parkin, 141C, and 321C, a RING1 specific antibody was used to detect parkin, 141C, and 141-409, and a Ubl specific antibody was used to detect parkin and Ubl. (D) Fluorescent images of PINK1 co-transformed with C-terminally YFP tagged parkin truncation variants grown in inducing liquid media for 16 hours were captured using a Leica TCS SP5 II confocal microscope at 63x magnification. (E) Western blot analysis of N2a cells transiently transfected with parkin truncation variant (141C), probed against parkin. Tubulin was used to assess equal loading for extracts. (F). Time course toxicity of N2a cells transiently transfected with parkin truncation variant (141C) and parkin were used to assess the effects of parkin alterations in mammalian cells. P value ≤ 0.01.
Additional file 1: Figure S6. Aggregation of purified parkin. SDD-AGE of pure parkin exposed to H2O2 under different condition detected by anti-parkin antibody.