PINK1 accumulates upon knockdown of Lon.

<p>(A) Western blot analysis of whole head homogenates from transgenic flies expressing a Myc-tagged PINK1 genomic construct and a UAS-RNAi construct driven by <i>elav-GAL4</i>. The RNAi constructs used were as follows: the non-specific control <i>mCherry</i>-RNAi (Control-R), <i>Lon</i>-RNAi1 (Lon-R1), and <i>Lon</i>-RNAi2 (Lon-R2). An anti-Myc antibody was used to detect PINK1 and an anti-Actin antibody was used as a protein loading control. (B) Densitometry of the PINK1-Myc bands from the indicated genotypes was performed using Fiji software. The PINK1-Myc band intensities were then normalized to their respective Actin loading controls, and these ratios were in turn normalized to the Control-R PINK1/Actin ratio. (C) Western blot analysis of Lon protease (Lon), Mitochondrial transcription factor A (TFAM), Heat shock protein 60 (Hsp60) and Actin in whole head homogenate from control and Lon deficient animals. (D) Quantification of the Lon band intensities from the indicated genotypes performed as described in B. (E) Quantification of the TFAM bands from the indicated genotypes performed as described in B. (F) Quantification of the Hsp60 bands from the indicated genotypes performed as described in B. (G) Flow cytometry analysis of dissociated cells from the brains of flies expressing the indicated RNAi constructs. The graph shows the normalized fluorescence intensity of TMRE, an indicator of mitochondrial membrane potential, relative to age-matched control animals. Fluorescence intensity was normalized to control samples prepared and analyzed on the same day as experimental samples. The mitochondrial membrane potential uncoupling agent CCCP was added to samples of the indicated genotypes to illustrate the effect of mitochondrial depolarization on TMRE signal intensity. All experiments described in this figure were repeated at least three times per genotype. Error bars represent the standard error of the mean (s.e.m.). *<i>p</i><0.05, **<i>p</i><0.005 by Student <i>t</i> test.