Fig. 4.

Citrate synthase and complex I activities and expression levels of single subunit proteins of respiratory complexes I–V in R349P desmin knock-in mice. a Determination of the citrate synthase activity by a spectrophotometric assay using identical amounts of total protein extracts. Note the marked activity reduction in homozygous mice. For this approach, soleus muscles obtained from five mice of each genotype (WT, HET, HOM) were pooled and subjected to four-time repeated measurements. Column chart shows mean values of these technical replicates. b Determination of complex I activity by a colorimetric assay normalized to muscle weight. Though this analysis, performed in duplicate on non-pooled samples derived from three animals per genotype, showed a reduction of complex I activity in homozygous mice, the data failed to reach statistical significance. An additional normalization of the obtained complex I values to the citrate synthase values resulted in similar activity levels for the three genotypes (data not shown). Column chart shows mean values and standard errors. c Immunoblotting using antibodies directed against specific complex I–V proteins revealed increased, decreased, and missing signals of complex I subunit proteins in heterozygous and homozygous mice, whereas the signal intensities of complex II–V subunit proteins showed no obvious differences. Desmin immunoblotting confirmed a markedly decreased level of mutant desmin in homozygous mice. In heterozygous mice the obtained signal is composed of the signals from both R349 wild-type and P349 mutant desmin, as previously demonstrated by high resolution SDS-PAGE and immunoblotting [16]. GAPDH immunoblotting and Coomassie stained SDS-PAGE gels were used as loading controls