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. 2014 Jan 6;23(10):2580–2592. doi: 10.1093/hmg/ddt652

Figure 3.

Figure 3.

Loss of LRPPRC is associated with the appearance of an oligomycin-insensitive subassembled ATP synthase complex. (A) The ATPase activity in the presence or absence of oligomycin (Oligo) was normalized to the activity in controls measured in the absence of oligomycin, The ATPase activity was determined in heart mitochondria from Lrpprc knockout and control mice at age 4 weeks. Open bars, control (n = 3); filled bars, knockout (n = 3). Error bars indicate means ± SEM. (B) The ATPase activity in the presence or absence of oligomycin (Oligo) was normalized to the activity in controls measured in the absence of oligomycin. The ATPase activity was determined in heart mitochondria from Lrpprc knockout and control mice at the age of 8 weeks. Open bars, control (n = 3); filled bars, knockout (n = 3). Error bars indicate mean ± SEM (*P < 0.05; **P < 0.01; ***P < 0.001). (C) The ATPase activity in the presence or absence of oligomycin (Oligo) was normalized to the activity in controls measured in the absence of oligomycin. The ATPase activity was determined in heart mitochondria from Lrpprc knockout and control mice at age 12 weeks. Open bars, control (n = 5); filled bars, knockout (n = 5). Error bars indicate means ± SEM (*P < 0.05; **P < 0.01; ***P < 0.001). (D) Blue native polyacrylamide gel electrophoresis analysis of heart mitochondria extracted with a ratio (1 g/g) of digitonin to mitochondrial protein from Lrpprc knockout and control mice at the age of 12 weeks. Immunodetection of COX II, ATPα, ATP8, OSCP and IF1 after transfer of proteins from the BN-PAGE to PVDF membrane. The position of supercomplexes (Sc), complex I (I), the ATP synthase monomer (V), ATP synthase subcomplexes (subVa, subVb), complex IV (IV) and complex II (II) are indicated on the left side. (E) The supra-molecular organization of ATP synthase was determined by clear native polyacrylamide gel electrophoresis (CN-PAGE) stained for in-gel ATPase activity (appear black after color inversion) without (right panel) or with oligomycin (left panel) in heart mitochondria from Lrpprc knockout and control at the age of 12 weeks. Mitochondria were solubilized with a ratio (1.5 g/g) of digitonin to mitochondrial protein (n = 6). The position of ATP synthase oligomers (Vx), dimer (V2), monomer (V1), subassemblies (subVa, subVb) and F1 are indicated on the left side. (F) Steady-state levels of different ATP synthase subunits analyzed by western blots analyses in heart mitochondria from Lrpprc knockout and control mice at age 12 weeks (n = 3).