Figure 3. Depletion of RBFA has no appreciable effect on mitochondrial protein synthesis.

(A) HEK293 cells were treated with RBFA (lanes 2 and 3) or non-targeting (NT, lanes 1 and 4) siRNA for 3 days and extracts (25 µg) were subjected to western blotting to compare OXPHOS (COX2, NDUFB8 and SDHA) or mitoribosomal (mS40, mS29 and uL3m) protein levels. (B) Following similar siRNA treatment (NT or RBFA-2) for 3 (lanes 1 and 2) or 6 (lanes 3 and 4) days, cells were metabolically labelled (³⁵S-met/cys) and extracts (3 days—30 µg; 6 days—50 µg) separated by 15% denaturing PAGE. Migration of the 13 mtDNA-encoded polypeptides is indicated and loading confirmed by Coomassie blue (CBB) staining.