Skip to main content
. 2018 Dec 20;38(2):e99916. doi: 10.15252/embj.201899916

Figure 3. BAK is ubiquitinated in response to various mitophagy stimuli and by endogenous Parkin, but is compromised in Parkinson's disease‐associated Parkin mutants.

Figure 3

  1. HeLa cells with or without HA‐Parkin were treated with CCCP (10 μM), antimycin A and oligomycin (AO) for 2 h or GTPP (10 μM) for 6 h. Immunoblots are representative of three independent experiments. Graph shows densitometric analysis of non‐ubiquitinated proteins relative to untreated control from three independent experiments. Error bars represent mean ± SD.
  2. SH‐SY5Y neuroblastoma cells expressing Cas9 together with a sgRNA targeting PRKN (sgPRKN) or a non‐targeting control (sgCon) were assessed for Parkin expression by immunoblotting of cytosolic fractions.
  3. SH‐SY5Y cells generated in (B) were treated with antimycin A and oligomycin (AO) for 3 h prior to cell lysis and immunoblotting. Mono and di‐ubiquitinated BAK in Parkin‐expressing cells following AO are indicated (*). Data are representative of three independent experiments.
  4. Domain architecture of human Parkin with positions of selected PD‐associated mutations. Ubl, ubiquitin‐like; IBR, in‐between‐RING (Gladkova et al, 2018). HeLa cells expressing wild‐type Parkin or the indicated PD‐associated Parkin mutants were treated with antimycin A and oligomycin (AO) for 2 h. Immunoblots are representative of three independent experiments. Graph shows densitometric analysis of non‐ubiquitinated proteins in AO‐treated samples relative to untreated control from three independent experiments. Error bars represent mean ± SD. All Parkin constructs were N‐terminally FLAG‐tagged except R104W.

Source data are available online for this figure.