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. 2012 Mar 9;287(18):14480–14493. doi: 10.1074/jbc.M111.322925

FIGURE 5.

FIGURE 5.

Expression, targeting, and assembly of C-terminal TAP-tagged TbTim17 in T. brucei mitochondria. C-terminal TAP-tagged-TbTim17 was expressed from the inducible vector pLew79-MHT. A, immunoblot analysis of total proteins from cells grown in the presence or absence of doxycycline for 48 and 96 h using antibodies against TbTim17. PAP reagent was used to detect TAP-tagged-Tim17. B, proteins from subcellular fractions were analyzed by immunoblot with antibodies against mitochondrial proteins VDAC and Tim17 and cytosolic protein PP5 and with PAP reagent. T, total fraction; C, cytosolic fraction; M, mitochondrial fraction. Ten micrograms of protein were loaded per lane. C, assembly of the C-terminal TAP-tagged TbTim17 into mitochondrial membrane protein complexes. Mitochondrial proteins from the parental, TbTim17 RNAi, and TbTim17-TAP cells grown for 48 h were solubilized with digitonin (1.0%) and analyzed by BN-PAGE and immunoblot using anti-TbTim17 and anti-VDAC antibodies. PAP reagent was used to detect TbTim17-TAP-containing protein complexes. Protein complexes and molecular size markers are indicated.