Figure 8.

An mtSSB-GRSF1 double knockdown shows an exacerbated phenotype with reduced steady-state ribosomal protein levels and evidence of reduced mitochondrial protein synthesis. Total cellular lysates (60 μg) were prepared from the same knockdown experiments used also for DNA isolation, RNA isolation and Northern blot analysis as shown in Figure 7. A representative Western blot is shown and probed with Twinkle, mtSSB and GRSF1 antibodies to illustrate the effect of the knockdown of these proteins (A). In addition, several other relevant antibodies were used such as mitoribosomal antibodies (MRPL3, MRPL49 and MRPS22), COX II and Suv3, as well as a mitochondrial loading marker (prohibitin 1, PHB) and a cellular marker (Actin). (B) The quantification of three biological repeats on 3 Western blots, for which the band intensities were determined and corrected for loading based on the actin signal. As for Figure 2 and 7, error bars show ± SEM. Statistics used one sample t-test comparing each sample to its own control that was set at 1. * indicates a P-value ≤ 0.05; ** indicates a P-value ≤ 0.01. The P value for the COXII band in the combined mtSSB/GRSF1 knockdown is separately indicated, as it just failed to reach statistical significance despite it being consistently down.