For the interaction between MITOSUR-Flag or MITOSURK513A-Flag and Mitok-V5, HeLa cells were transfected with the indicated plasmids. After 48 hours, cells were lysed in CoIP buffer (150mM NaCl, 1% Digitonin, 50mM Tris-Cl pH7.4, 1mM EGTA-Tris pH 7.4 and Complete EDTA-free protease inhibitor mixture). Lysates were centrifuged at 15000xg for 10 min, and supernatant was transferred into new tubes. One mg of proteins was precleared using a control agarose resin (Thermo Fisher Scientific) for 30 min at 4°C. Precleared proteins were incubated with monoclonal α-Flag-agarose-conjugated antibody (Sigma) for 3 hours at 4°C. After 3 washes (10’) in CoIP buffer, 50 μl of Laemmli buffer 2X was added to the resin and heated for 5 min at 95°C. The precleared lysate (Input) and the immunoprecipitated (CoIP) fractions were separated and blotted as described above.
For the interaction between endogenous MITOK and MITOSUR, isolated mitochondria from mouse liver or HeLa cells were lysed in CoIP buffer and processed as indicated above. One mg of precleared proteins were incubated with 5 μg of anti-MITOKN-term (Sigma HPA010980) antibody for 3 hours. Protein A-Sepharose beads (GE Healthcare) were added for 1 hour and washed 3 times with CoIP buffer. 50 μl of Laemmli buffer 2X was added to the resin and heated for 5 min at 95°C. Results are representative of at least three independent transfections.
For the interaction between endogenous MITOK and MITOSUR, isolated mitochondria from mouse liver or HeLa cells were lysed in CoIP buffer and processed as indicated above. One mg of precleared proteins were incubated with 5 μg of anti-MITOKN-term (Sigma HPA010980) antibody for 3 hours. Protein A-Sepharose beads (GE Healthcare) were added for 1 hour and washed 3 times with CoIP buffer. 50 μl of Laemmli buffer 2X was added to the resin and heated for 5 min at 95°C. Results are representative of at least three independent transfections.