Supplementary Figure 4: Lys6 ubiquitin chains are enriched in HEK293 and SH-SY5Y whole cell lysates and mitochondria upon CCCP treatment.

(A) SH-SY5Y Western blot confirmation of mitochondrial depolarization upon CCCP treatment over time. MFN1 levels decrease and Opa1 processing increased over the course of 4 h with 10 μM CCCP treatment in SH-SY5Y cells. (B) Base peak chromatograms of immunoaffinity-purified K-GG peptides from SH-SY5Y cells following Parkin overexpression (oe) and/or 10 μM CCCP treatment for 2 h. Chromatographic features showing ubiquitin signature peptides for Lys6, Lys11, Lys48, and Lys63-linked chains are denoted by arrows. Inset shows extracted ion chromatograms from Lys6 peptides in the treated and untreated samples. (C) SDS-PAGE gel regions excised for ubiquitin linkage analysis in Supplementary Fig. 1D. HEK293 cells stably expressing GFP-Parkin were treated with either DMSO or 20 μM CCCP for 3 h, fractionated into mitochondria, and lysed for trypsinization and subsequent ubiquitin linkage analysis. Data represent one out of three independent experiments. (D) Ubiquitin linkage analysis on 4 gel regions ranging from 5–250 kDa. Abundance (fmol) of each ubiquitin linkage is reported for DMSO (black circles) or CCCP (red circles) treatment for each gel region. Data represent one out of three experiments. (E) Representative SDS-PAGE gel showing excised regions for ubiquitin linkage analysis in Supplementary Fig. 1F. SH-SY5Y cells were treated with either DMSO or 10 μM CCCP for 3 h, fractionated into cytosolic and mitochondrial fractions, and lysed for trypsinization and subsequent ubiquitin linkage analysis. (F) Ubiquitin linkage analysis of cytosolic and mitochondrial fractions of 2 gel regions ranging from 40–100 kDa. Abundance (fmol) of each ubiquitin linkage is reported for DMSO (black circles) or CCCP (red circles) treatment for each gel region from either the cytosolic or mitochondrial fractions of two replicate experiments in SH-SY5Y cells.