Assembly of fusion proteins into mitochondrial complexes in HeLa cells was tested by in-gel fluorescence after 2-D BN/SDS-PAGE.

GFP and Dendra2 fluorescence was preserved after SDS-PAGE and signals are shown in grayscale (lower panels). A. The same gels were silver-stained. The gel of CIV-G expressed HeLa cells was shown as an example. The positions of the fusion proteins were marked (left axis). B. Complex I – 30 kDa-GFP (CI-G) was detected in complex I, in supercomplexes (S–G) and in subcomplexes of complex I. C. Complex II-subunit B-GFP (CII-G) was predominantly found in complex II. D. Complex III-6.4 kDa subunit -GFP (CIII-G) was predominantly found in the dimer of complex III (CIII2-G), additionally in small and large supercomplexes (S–G and III2IV1-G). E. Complex IV-Cox8a-GFP (CIV-G) was detected predominantly in complex IV, and smaller amounts were assembled into supercomplexes (S–G and III2IV1-G). F. Complex V-γ-Dendra2 (CV-D) was detected in holo complex V (V–D) and, in F1 subcomplexes (F1-D). At the gel front, individual fusion proteins (*) together with degradation products (**) were detected (B–D, F). S, large supercomplexes composed of respiratory chain complexes I, III2, and IV; III2IV1, small supercomplexes composed of respiratory chain complexes III2, and IV; V, complex V or ATP synthase; IV, complex IV or cytochrome c oxidase; III2, dimer of complex III or cytochrome c reductase; II, complex II; F1, subcomplex of complex V; G, GFP; D, Dendra2, BNE, Blue native electrophoresis.