Journal: Autophagy

Article Title: Phospho-ubiquitin-PARK2 complex as a marker for mitophagy defects

doi: 10.1080/15548627.2016.1254852

Figure Lengend Snippet: PARK2 forms a phospho-ubiquitin-rich complex with VDAC. (A) FLAG-PARK2 and control cells were differentially labeled using SILAC. Equal amounts of mitochondria were mixed, solubilized and subjected to anti-FLAG immunoprecipitation, followed by native elution. Complexes from 2 independent experiments (with label switch) were resolved using BN-PAGE. Gel lanes were cut into equal slices, within the range of the 500-kDa complex, and each slice was analyzed by LC-MS. Shown are relative protein abundance ratios (FLAG-PARK2:control). (B) Solubilized mitochondria from FLAG-PARK2 cells treated with CCCP (3 h) were analyzed by 2D BN/SDS-PAGE, followed by western blotting. Asterisk indicates likely ubiquitinated forms of VDAC. (C) Anti-FLAG immunoprecipitations using mitochondria solubilized from FLAG-PARK2-expressing cells, treated with 10 μM CCCP, or with DMSO as a control, for 3 h. Solubilized mitochondria from COX6A1-FLAG-expressing cells were used as a specificity control.26 Eluates were analyzed by SDS-PAGE, followed by western blotting using antiserum for the indicated proteins. Total, 1%; Eluate, 100%. (D) Immunoprecipitated FLAG-PARK2 complexes from CCCP (3 h)-treated cells were analyzed by 2D gel analysis as in (B). Asterisk indicates likely ubiquitinated forms of VDAC. (E) Immunoprecipitated FLAG-PARK2 complexes from mitochondria, after CCCP or DMSO treatment of cells, were incubated with the deubiquitinase USP2, or the buffer control, for 1 h at 37°C. Samples were split and analyzed by BN-PAGE (right panel) and SDS-PAGE (left panel), followed by western blotting with the indicated antibodies. (F) Mitochondria from FLAG-PARK2 cells treated with CCCP for 3 h were solubilized in buffer containing 2% anti-VDAC serum or the respective pre-immune serum (control). Following solubilization, protein complexes were resolved by BN-PAGE analysis (4-13%) and were subsequently immunoblotted. (G) Mitochondria from wild-type cells treated with 10 µM CCCP, or DMSO control, for 3 h were solubilized and analyzed as in (B).

Article Snippet: The deubiquitinase USP2 was purchased from BostonBiochem® (E-506).

Techniques: Labeling, Immunoprecipitation, Liquid Chromatography with Mass Spectroscopy, SDS Page, Western Blot, Expressing, Two-Dimensional Gel Electrophoresis, Incubation