Endogenous coimmunoprecipitation

Wash Protein G agarose fast-flow beads in TBS 1X + 0.05% NP40.To this

end, the beads were incubated with lysis buffer on the spinning wheel (25 RPM) for 2

–5 min at 4 °C, followed by a 1 min centrifugation at 500 RPM 4 °C.

Repeat step 1 for a total of 3 washes.

Incubate 20ul of pure beads with 5ug of anti-LONP1 antibody or 5ug of normal rabbit IgG as control (or 3ug of anti-Hsp60 and 3ug of mouse IgG as control) and add 300ul of washing buffer to each tube.

Incubate 2h at 20RPM on a rotating wheel at 4°C

Meanwhile, detach HEK cells using Accutase for 5 minutes at 37°C and collect them.

Detach cells using Accutase for 5 minutes at 37°C and collect them.

Spin cells in a centrifuge at 250g for 5 minutes at room temperature.

Remove the supernatant and wash cells in PBS.

Repeat steps 7 and 8 for a total of 2 washes.

Lyse cells in 1% TBS + 0.5% NP40 + PI/PHI (Pierce #A32959)

After 2h incubation, wash beads again 3 times in washing buffer.

Incubate antibody-coated beads with 3.7 ug of lysate on the spinning wheel for 2h.

After 2h incubation, wash beads again 3 times in washing buffer.

Elute by boiling the beads twice with 2x Laemmli buffer at 95°C for 8min at 300rpm in a thermoblock.

Spin beads for 1 minute at 10000 RCF.

Collect supernatant and proceed with western blot analysis.