CoxII degradation assay to assess mitophagy

Seed the hela cells the day before the treatment day in 6 well plates.

Each well contain 2mL of growth media.

Seed 350,000 cells for penta KO expressing BFP-Parkin and GFP-OPTN or -NDP52.

Adjust the number of cells of other cell lines. So that the next day they are all in similar confluency with penta KO expressing BFP-Parkin and GFP-OPTN or -NDP52.

The next day, make sure the seeded cells are spreading out (not concentrated in the middle of the well because this can affect the results).

Aspirate off the old media and treat each well with 2mL of growth media containing 4micromolar (µM) Antimycin A, 10micromolar (µM) Oligomycin and 10micromolar (µM) QVD for desired period.

After the treatment, harvest the cells on ice by scraping.

Pre-chill eppies and 1x PBS on ice.

Aspirate off all the PBS.

Aspirate the media thoroughly from the wells.

Wash the wells with 1mL of cold 1x PBS.

Aspirate off the PBS and add another 1mL of cold 1x PBS.

Use a plastic cell scraper to scrape all the cells off the wells.

Transfer the cells-containing PBS to eppies.

Centrifuge the eppies at 3000rpm,0h 0m 0s for 0h 2m 0s at 4°C.

Aspirate off PBS.

Quickly centrifuge for 0h 0m 10s to spin down the residual PBS.

Lyse the cell pellets in lysis buffer and boil the samples at 99°C with shaking for 0h 7m 0s.

Let the samples cool down and spin at max speed (Room temperature) for 0h 1m 0s.

Estimate the protein concentration by nanodrop

Aliquot 25µgof each sample into a new eppie and add 1x LDS to make up to 15µL.

Set up the gel tank with MOPs buffer.

Wash each well with a glass syringe.

Load markers and samples into the wells and run at 100V for 0h 10m 0s and 190V for 0h 55m 0s.

Subject the gels to wet transfer onto PVDF membrane using cold NuPAGE transfer buffer containing 20% Methanol for 1h 0m 0s at Room temperature.

After transfer, incubate the PVDF membrane with PVDF destain buffer on a shaker at Room temperature for 0h 2m 0s.

Wash three times with PBS/Tween ( 5 min each wash).

Wash the PVDF membrane with PBS/Tween for 0h 5m 0s (1/3).

Wash the PVDF membrane with PBS/Tween for 0h 5m 0s(2/3).

Wash the PVDF membrane with PBS/Tween for 0h 5m 0s(3/3).

Block with blocking buffer for 0h 15m 0s.

Remove blocking buffer.

Rinse twice with PBS/Tween and wash twice with PBS/Tween and once with 1x PBS (5 min for each wash).

Rinse the blocking buffer with PBS/Tween and wash with PBS/Tween for 0h 5m 0s(1/2).

Rinse the blocking buffer with PBS/Tween and wash with PBS/Tween for 0h 5m 0s(2/2).

Rinse the blocking buffer with 1x PBS and wash with 1x PBS for 0h 5m 0s.

Cut the PVDF membrane and put appropriate parts into different antibodies.

Incubate on a 4°C shaker .

The next day, recycle the antibodies back to their tubes.

Wash the blots three times with PBS/Tween (5 min for each wash).

Wash the blot with PBS/Tween for 0h 5m 0s(1/3).

Wash the blot with PBS/Tween for 0h 5m 0s(2/3).

Wash the blot with PBS/Tween for 0h 5m 0s(3/3).

Incubate with appropriate HRP-conjugated secondary antibodies made up in blocking buffer for 1h 0m 0s.

Wash the blots twice with PBS/Tween, once with 1x PBS (5 min for each wash).

Wash the blot with PBS/Tween for 0h 5m 0s (1/2).

Wash the blot with PBS/Tween for 0h 5m 0s (2/2).

Wash the blot with 1x PBS for 0h 5m 0s .

Develop the blots with ECL prime.