BN-PAGE on human fibroblasts were performed using NativePAGE reagent (Invitrogen) according to the standard procedure. Briefly, cells were lysed 15 min on ice in NativePAGE sample buffer 1X containing 1% digitonin or 2% digitonin for the extraction of PHB or MICOS complex, respectively. The lysate was clarified by centrifugation at 20,000xg for 30min at 4°C. Fifteen micrograms of proteins were separated by Blue Native-PAGE Novex 4-16% Bis-Tris gel (Thermo Fisher Scientific). Gels were stained with Coomassie Brilliant Blue.
Proteins from the resulting gels were transferred to PVDF membranes (Millipore), and analyzed by western blotting with relevant antibodies. BN-PAGE on mouse tissues were performed with 15g of mitochondrial respiratory chain complexes obtained by solubilization in a solution of 1.5M aminocaproic acid (Sigma-Aldrich), 75mM Bis-TRIS (Sigma-Aldrich) and 4% dodecyl-b-D-maltoside (Sigma-Aldrich), were separated by Blue Native-PAGE Novex 4-16% Bis-Tris gel (Thermo Fisher Scientific). Samples were then electroblotted onto a PVDF membrane before sequential incubation with specific antibodies allowing to verify that samples were equally loaded between mutants and controls.
Proteins from the resulting gels were transferred to PVDF membranes (Millipore), and analyzed by western blotting with relevant antibodies. BN-PAGE on mouse tissues were performed with 15g of mitochondrial respiratory chain complexes obtained by solubilization in a solution of 1.5M aminocaproic acid (Sigma-Aldrich), 75mM Bis-TRIS (Sigma-Aldrich) and 4% dodecyl-b-D-maltoside (Sigma-Aldrich), were separated by Blue Native-PAGE Novex 4-16% Bis-Tris gel (Thermo Fisher Scientific). Samples were then electroblotted onto a PVDF membrane before sequential incubation with specific antibodies allowing to verify that samples were equally loaded between mutants and controls.