Cells were grown in 6-well dishes (6.0 × 105 cells/well) for 24 h. Then, 100 μg/mL cycloheximide (CHX) was added, and the cells were collected at 0, 6 and 12 h after addition. Collected samples were lysed with lysis buffer, and western blot analysis was performed. Proteasome inhibition was performed using cells treated with either 20 μM DMSO or 20 μM MG132 (Sigma) for 12 h. Cellular lysate or mitochondrial proteins were then isolated and subjected to western blotting. Ubiquitin accumulation in WT and MTU1 mutants was determined using a K48-Ubiquitin antibody (Cell Signaling Technology).
For evaluation of MTU1 protein stability in CLPP-, LONP1- and OMA1-knockdown cells, cells expressing WT or mutant MTU1 were transfected with 20 μM of predesigned Silencer® Select validated siRNA targeting CLPP (ID# s15688, ID# s15686), LONP1 (ID# s17903, ID# s17902) or OMA1 (ID# s41776, ID#s41777) and Silencer Select Negative control #1 (Invitrogen) using Lipofectamine RNAiMAX Transfection Reagent (Invitrogen) according to the manufacturer's instructions. After 48 h, transfected cells were then treated with 100 μg/mL CHX for 0, 6 and 12 h, and knockdown efficiency and protein stability were determined by western blotting analysis.
For evaluation of MTU1 protein stability in CLPP-, LONP1- and OMA1-knockdown cells, cells expressing WT or mutant MTU1 were transfected with 20 μM of predesigned Silencer® Select validated siRNA targeting CLPP (ID# s15688, ID# s15686), LONP1 (ID# s17903, ID# s17902) or OMA1 (ID# s41776, ID#s41777) and Silencer Select Negative control #1 (Invitrogen) using Lipofectamine RNAiMAX Transfection Reagent (Invitrogen) according to the manufacturer's instructions. After 48 h, transfected cells were then treated with 100 μg/mL CHX for 0, 6 and 12 h, and knockdown efficiency and protein stability were determined by western blotting analysis.