Figure 2.
Reduced Complex IV Activity and Amount in Fibroblasts from the Patients with a C2orf64 Mutation
(A) Fibroblasts from patients P1 and P2 show a severely reduced in-gel activity of complex IV32 compared to the unaffected siblings S3 and S4 (top panel). The lower three panels show the results of immunoblots after nondenaturing BN-PAGE,33 revealing a severely reduced complex IV amount in patients P1 and P2. Complex IV was stained with anti-COX4 antibodies. Equal loading of the gel was tested by staining for complex III (by using anti-CORE2) and complex II (by using anti-70 kDa subunit). Holocomplex IV is indicated by the arrowhead; the asterisk indicates a nonspecific band.
(B) Immunoblot after SDS-PAGE of fibroblast extracts of patients P1 and P2 and healthy siblings S3 and S4 showing reduced expression levels of complex IV subunits COX1, COX2, COX4, and COX5a in fibroblasts of both patients. The complex II 70 kDa subunit (CII 70 kDa) was used as a loading control.
(C) Two dimensional BN-PAGE analysis of fibroblasts from patients P1 and P2 and the healthy siblings S3 and S4 was performed in accordance with standard procedures. Blots were stained by using antibodies against different complex IV subunits as indicated. Holocomplex IV is indicated (H). Patients P1 and P2 show accumulation of a small subcomplex (indicated by S1) that contains COX1 but not COX2, COX4, and COX5a. This indicates that mutation of C2orf64 results in accumulation of a COX1 containing early complex IV assembly intermediate. Note that blots of different fibroblasts and blots stained with different antibodies can only be compared qualitatively because exposure times are not the same.