Sc 100496

Immunoblots were performed as previously described [5 (link)]. Antibodies against p16 (1:200 dilution; #sc-1207), ALDH2 (1:200 dilution; #sc-100496) and acetylated FoxO1 (1:200 dilution; #sc-49437) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against p53 (1:1000 dilution; #2524), LC3B (1:1000 dilution; #3868), SirT1 (1:1000 dilution; #3931), acetyl-lysine (1:1000 dilution; #9441), Atg7 (1:1000 dilution; #8558), Rab7 (1:1000 dilution; #9367) and FoxO1 (1:1000 dilution; #2880) were purchased from Cell Signaling Technology. Antibody against DNPH (1:2000 dilution; #ab93160), LAMP2 (1:1000 dilution; #ab25339), Tubulin (1:1000 dilution; #ab179513), GAPDH (1:10000 dilution; #ab181602) and TBP (1:5000 dilution; #ab28175) were purchased from Abcam. Antibody binding was detected via enhanced chemiluminescence (Millipore) and scanned with ChemiDocXRS (Bio-Rad Laboratory, Hercules, CA). Immunoblot band intensity was analyzed with Lab Image software. For immunoprecipitation analysis, lystes were mixed with primary antibody. Immunocomplexes were separated by SDS-PAGE and detected with western blot analyses.

A mitochondrial PPI network was generated by retaining interactions from two or more published studies (PMID are listed in Supplementary Table 5 for each interaction) for proteins whose level was altered in PARK2-mutated patients. The network was then visualized using Cytoscape.
A small set of interesting candidate interactions of physiological relevance was confirmed in control subject C4 and PARK2-mutated PD patient P3 by immunoprecipitation. Mitochondrial pellets were cross-linked with 1 mM dithiobis (succinimidyl propionate) (DSP; Thermo Fisher Scientific) and lysed with 500 μl RIPA buffer. Inputs were controlled by western blotting. Three microliter of each antibody [HSPA8, abcam (ab51052); HSPD1, abcam (ab46798); ALDH2, Santa Cruz Biotechnology (sc-100496)] were added to 1 mg of mitochondrial protein lysates, which were then incubated for 1 h in agitation at 4°C. One hundred microliter of μMACS magnetic microbeads (Miltenyi) were then added to lysates and incubated with continued agitation ON at 4°C. After washing the microbeads suspensions using 0.1% RIPA buffer through μMACS columns (Miltenyi), proteins were eluted using 200 μl of Laemmli buffer, previously heated at 95°C. Eluates were analyzed by western blotting.