Fig. 4. Loss of POLRMT results in an increased mtDNA-free pool of TFAM.

(A) Steady-state protein levels of nuclear-encoded factors of mtDNA expression analyzed by Western blotting on mitochondrial extracts from hearts of control and tissue-specific knockout mice; loading, VDAC; asterisk, cross-reacting band (28); for quantification, see fig. S4A. (B) Quantitative RT-PCR (qRT-PCR) of transcript levels of nuclear-encoded mitochondrial proteins. Normalization, β₂M (β₂-microglobulin). Error bars indicate ±SEM (*P < 0.05 and ***P < 0.001; two-tailed Student’s t test; see table S1). (C and D) Linear glycerol density gradient fractionations of mitochondrial lysates from tissue-specific knockout and control mice followed by Western blot analysis; for quantification, see figs. S5 (A to C) and S6. Samples taken from fractions 1 to 16 are of increasing density (that is, from top to bottom of the tube after separation by ultracentrifugation; as indicated by the schematic representation of the centrifuge tube to the left). Fractions were loaded from left to right on the gels as indicated by the lane numbering; input, aliquots of unfractionated lysates. The mtDNA content of the fractions was determined by Southern blotting. (E) Relative TFAM and POLRMT protein distribution across the gradient from control and knockout heart mitochondria.