Supplementary Figure 7: Mass spectrometry validation and ubiquitin linkage analysis of USP30 knockout cell line.

(A,B) Mass spectrometry confirmation of USP30 CRISPR knockout. Ratios of area under the curve (AUC) between USP30 knockout HEK293 cells expressing GFP-Parkin and a wild type USP30 isogenic cell line for 2 mitochondrial matrix proteins (HSPD1, ATP5B) and three independent USP30 peptides (A; see Supplementary Table 2 for raw data). Ratios are not shown for the three USP30 signature peptides since no signal was detected in the USP30 knockout cell line, as represented in (B). Error bars represent standard deviation from n = 3 independent experiments (Supplementary Table 2). (C) Representative SDS-PAGE gel from three independent experiments showing excision locations used for ubiquitin linkage analysis in Fig. 3a and Supplementary Fig. 3D. USP30 knockout (KO) or WT USP30 (WT) HEK293 cells stably expressing GFP-Parkin were treated with either DMSO or CCCP (10 μM, 3 h), enriched for mitochondria, and lysed for trypsinization and subsequent ubiquitin linkage analysis. (D) Ubiquitin linkage analysis on 4 gel regions ranging from 5–250 kDa. fmol of each ubiquitin linkage is reported for CCCP treated WT (black circles) or USP30 knockout (red circles) mitochondrial enriched fractions for each gel region. Data represent one out of two replicate experiments. (E) Representative SDS-PAGE gel showing regions excised for ubiquitin linkage analysis in Fig. 3c.