NT-OPA1-exon4b is embedded in the inner mitochondrial membrane and interacts with the nucleoid. (A, left) Western blot showing the presence of OPA1 and NT-OPA1-exon4b in the pellet (P) or supernatant (SN) fractions of mitochondria, either untreated, or treated with 1 M NaCl or 3% SDS. (Right) Western blot showing the presence or absence of OPA1, HSPD1, and NT-OPA1-exon4b in mitochondrial fractions treated successively with trypsin, digitonin, and triton. (B, left) Western blots using TFAM, POLG, and VDAC1 antibodies, performed on the flow-through (Ft) and eluted (El) fractions from immunoprecipitated mitochondrial proteins purified from mocked cells or cells transfected with plasmids expressing Flag-tagged TFAM, NT-OPA1-exon4, or NT-OPA1-exon4b, evidence the specific presence of TFAM and POLG but not VDAC1, in the eluted fractions of cells specifically expressing the TFAM and NT-OPA1-exon4b proteins. (Right) Measurements of mtDNA recovery, as the ratio between the amounts of mtDNA in the immunoprecipitates from that in the input.
