Bn page

To identify and estimate the levels of respiratory supercomplexes, homogenates from skeletal muscle were treated with digitonin (ratio 1:8, protein: digitonin, Roche) and mitochondrial complexes separated by blue native PAGE (BN‐PAGE) in 3–12% acrylamide gradient gels (Invitrogen; Wittig et al, 2006; Diaz et al, 2009). Protein (10–20 μg) was used and separated by PAGE, transferred to a PVDF membrane (Bio‐Rad), and incubated sequentially with antibodies against several subunits of the different mitochondrial respiratory chain complexes. To detect the activity of complex I in‐gel, samples (60 μg) treated with digitonin were separated in 3–12% gels and incubated with 0.14 mM NADH and 1 mg/ml of nitroblue tetrazolium in Tris–HCl 0.1 M pH 7.4. The gels were incubated at 37°C for approximately 1 h. To detect the activity of complex IV, the gels were incubated with 5 mg of diaminobenzidine (DAB) dissolved in 9 ml of 50 M phosphate buffer pH 7.4 containing 10 mg cytochrome c and 750 mg of sucrose. The gels were incubated at 37°C until a brown color was observed.

Typically, equal amounts of YaxA and YaxB were combined and incubated at 25 °C for 30 min. Cymal-6 (1.5% w/v) was added and the mixture was incubated at 4 °C for 30 min, then injected onto a Superose 6 column running in the gel filtration buffer (25 mM HEPES pH 7.0, 150 mM NaCl, 0.05% w/v Cymal-6). To separate different oligomeric states of protein complexes, peak fractions were separated by 4-16 % gradient blue native polyacrylamide gel electrophoresis (BN-PAGE; Thermo Fisher Scientific Korea, Invitrogen^TM, South Korea) without any other additives. The bands corresponding to YaxAB-C₈, -C₉, and -C₁₀ pores were cut out from the gel, soaked in the gel filtration buffer, and then incubated at 4 °C overnight to extract the proteins. The supernatant containing oligomeric YaxAB was used for the following experiments.

H1299 cells were transfected using the Qiagen Effectene kit according to the manufacturer’s manual in a 10 cm dish, grown for about 24 h, harvested and collected in NP lysis buffer without detergent (20 mM Tris, 150 mM NaCl, 2 mM MgCl₂, 1 mM DTT, complete protease inhibitor cocktail (Roche), PhosSTOP (Roche) pH 7.4). Samples were lysed via three freeze and thaw cycles. After centrifugation, Benzonase (Merck Millipore) was added and incubated 1 h on ice. Samples were split and incubated with different urea concentration for 1 h on ice. An equal volume of NP lysis buffer was added containing the two-fold concentration of CHAPS (20 mM Tris, 150 mM NaCl, 2 mM MgCl₂, 40 mM CHAPS, 1 mM DTT, complete protease inhibitor cocktail (Roche), PhosSTOP (Roche) pH 7.4) and incubated for about 10 min. Afterwards, 3× NP sample buffer (60% Glycerol, 15 mM Coomassie G250) was added and the sample was loaded on the gel. BN-PAGE (3–12%, Thermo Fisher Scientific) was performed and blotted as previously described^{13 (link),35 (link)}.

H1299 cells were transfected using the Qiagen Effectene kit according to the manufacturer’s manual in a 12-well plate or H1299 cells stably expressing p63 isoforms were used. Cells were harvested and lysed in NP lysis buffer (20 mM Tris, 150 mM NaCl, 2 mM MgCl₂, 20 mM CHAPS, 1 mM DTT, complete protease inhibitor cocktail (Roche), PhosSTOP (Roche) pH 7.4 or pH 8.0). Benzonase (Merck) was added and samples were lysed on ice for 1 h. BN-PAGE (3–12%, Thermo Fisher Scientific) was performed and blotted as previously described^{17 (link),35 (link)}. Protein levels loaded on BN-PAGE were adjusted to equal p63 amounts by prior western blot analysis. SDS-PAGE to detect phosphoshifts were not input adjusted.