Anti hsp60

To measure mitochondrial protein glutathionylation, mitochondrial lysates were separated by SDS-PAGE. Blots were probed for glutathionylation using a 1:1,000 dilution of the anti-GSH antibody (Virogen, Watertown, MA) followed by a 1:1,000 dilution of horseradish peroxidase-linked goat anti-mouse Ig secondary antibody (Cell Signaling). Blots were also probed with a 1:5,000 dilution of anti-Hsp60 (AbCam, Cambridge, MA) to control for equal loading. To visualize multiple bands on the same blot, blots were stripped with Restore Western Blot Stripping Buffer (Pierce) before being probed with a new antibody. Proteins were normalized to Hsp60 to correct for loading differences. To determine the overall level of protein glutathionylation, bands were quantified using a ScanJet 4C scanner (Hewlett Packard, Palo Alto, CA) and Image J analysis software (National Institutes of Health, Bethesda, MA). To measure Complex III protein glutathionylation, mitochondrial cell lysates were immunoprecipitated for Complex III using a Complex III Immunocapture kit (AbCam), separated by SDS PAGE and immunobloted with the anti-GSH antibody, as previously described [11 (link)].

Anti-Myc (9E10) (Cat. Sc-40), anti-Ki67 (Cat. ab15580) and anti-Hsp60 (Cat. ab46798) antibodies were purchased from Abcam (UK); anti-β-actin (Cat. 4970S), anti-Cleaved caspase-3 (Cat. 9661S), and anti-Nur77 (Cat. 3960S) antibodies were purchased from Cell Signal Technology (Beverly, MA, USA); anti-Nur77 (M-210) (Cat. sc-5569), anti-PCNA (Santa Cruz sc-7907), anti-a-tublin (Santa Cruz sc-8035), anti-Bcl-2 (Santa Cruz sc-783), anti-PARP (Santa Cruz sc-7150), and anti-GST (sc-138) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-Flag (Cat.F1804) antibody was purchased from Sigma (St. Louis, MO, USA); Mito-tracker deep red (Cat. M22426), JC-1 Probe (Cat. T3168) and mitoSOX Red Mitochondrial Superoxide Indicator (Cat. M36008) were purchased from Thermo Fisher.

PAC MV^D7 stable cells were plated on glass bottom plates (Mattek) and grown overnight. The following day, cells were fixed in 4% paraformaldehyde in PBS for 10 min at room temperature. Cells were briefly rinsed with PBS and blocked/permeabilized in 5% Donkey serum + 0.1% Triton X 100 + PBS for 1 h at room temperature. Blocking buffer was replaced with primary antibodies (mouse anti-Hsp60 1:100, rabbit anti-mCherry 1:500, abcam) diluted in primary antibody buffer containing 1% BSA + 0.1% Triton X 100 in PBS and cells were incubated in primary antibody overnight at 4 °C. The following day primary antibody was aspirated, and cells were washed 3 × 5 min in PBS. Secondary antibodies (anti-mouse Alexa Fluor 488 and anti-rabbit Alexa Fluor 568, ThermoFisher) were applied in blocking buffer at a dilution of 1:1000 and incubated at room temperature for 1 h. Cells were washed once with PBS for 5 min before staining with Alexafluor 647-conjugated phalloidin (1:100) in 1% BSA in PBS for 15 min. Cells were washed and stained with Hoechst 33342 (5 μg/mL). Cells were imaged on an inverted Olympus FV1000 confocal microscope using a 60X oil immersion objective.
Colocalization analysis of OMM-PAC cells. Images of OMM-PAC cells prepared as described above were analyzed using imageJ’s Coloc2 plugin with Costes thresholding.