Fig. 6. LONP1 forms a complex with the mitochondrial protein import machinery.

HEK293 cells carrying inducible plasmids expressing 3× FLAG-tagged wild-type LONP1 were cultured for 3 d in the presence of 1 ng/mL doxycycline. The cells were extracted with RIPA buffer after treatment with the reversible crosslinker (DSP). The supernatant was recovered as the total protein (Total) following sonication and centrifugation. The FLAG-tagged LONP1 protein was immunoprecipitated using anti-FLAG-tag mAb-magnetic beads. After magnetic separation, unbound proteins were recovered as the flow-through (FT). The magnetic beads were incubated twice with RIPA buffer containing FLAG peptides and recovered as eluate (Elute). After cleavage of a disulfide bond in DSP, all samples were analyzed by immunoblotting. The experiments were repeated three times and representative data are shown.