Supplementary Figure 5: Immunodepletion of GFP-Parkin confirms substrates other than Parkin carry Lys6-linked polyubiquitin chains.
From: USP30 and parkin homeostatically regulate atypical ubiquitin chains on mitochondria
(A) HEK293 cells overexpressing GFP-Parkin were treated with 10 μM CCCP or a DMSO vehicle control for 2 h. Lysates were immunodepleted with anti-GFP beads and input and flow through (FT) fractions were run on Western blot. MFN1 levels decreased and Opa1 processing increased confirming mitochondrial depolarization upon CCCP treatment. This experiment was performed once. (B) Input and flow through (FT) fractions treated with CCCP from (A) were run on SDS-PAGE and excised into 10 gel regions spanning 5–250 kDa. (C) Ubiquitin linkage analysis was used to measure the abundance of Lys6 chains (fmol) for each gel region excised (left) and Parkin peptide abundance (AUC) was measured for each gel region excised (right).
