FIGURE 1

Y955C TG Pol protein expression and gene copy dosage: Protein from purified mitochondrial lysates of TG and control hearts were separated on a 4–12% polyacrylamide gel and either transferred to Immobilon-P for Western blot analysis or Coomassie Blue staining. (a) Western blot analysis of the heart mitochondrial extracts. Lane 1, purified recombinant human Pol as control; lanes 2, 4, and 6, mitochondrial lysates from Y955C TG hearts of 33-, 25-, and 10-week-old mice, respectively. Lanes 3, 5, and 7, mitochondrial lysates from control TG-negative hearts of 33-, 25-, and 10-week-old mice, respectively. Arrow indicates pol . (b) Coomassie Blue-stained gel to demonstrate that equivalent portions of heart mitochondrial proteins were analyzed by Western blot. Lanes 1–7 are identical to that in (a) except that five times the amount of protein was loaded and separated in the gel for detection by Coomassie Blue staining. (c) Relative gene copy number of mutant Y955C Pol was determined from cardiac tissues for each of the four lines generated. Arrow indicates pol . (c) Relative gene copy number of human mutant Y955C Pol was determined from cardiac tissues for each of the four lines generated. Levels of POLG were analyzed semiquantitatively from murine tail DNA extracts using real-time PCR and Light Cycler TaqMan Master kit and normalized to GAPDH from WT.