Figure 4.
Knockdown of C12orf62 in Control and Subject Fibroblasts Results in a Specific Defect in COX Assembly and COX I Translation
(A) BN-PAGE analysis of the assembly of individual OXPHOS complexes in control and subject fibroblasts transiently transfected with two different siRNA constructs specific to C12orf62 (KD1 and KD2), with a fluorescent control siRNA (Alexa), and without siRNA (Mock). Complex IV (OE) is a longer exposure of the same blot.
(B) Pulse labeling with [35S] methionine and [35S] cysteine of mitochondrial polypeptides of the samples in panel (A). The seven subunits of complex I (ND), one subunit of complex III (cyt b), three subunits of complex IV (COX), and two subunits of complex V (ATP) are indicated at the left of the figure. For quantification of COX I synthesis, COX I labeling was normalized to the labeling of ND3. COX activity was also measured in these samples. The values are shown under the translation gel and are expressed as percentages of the mock control.
(C) The mitochondrial translation products were pulse labeled and chased for 10 min (PULSE) or 17 hr (CHASE) in control and subject fibroblasts and in cells that were transfected with either a siRNA construct specific to C12orf62 (KD2) or a fluorescent control siRNA (Alexa). COX I labeling was normalized to the labeling of ND3.