Figure 2.

mPTP activation increases C-subunit content of the channel-forming fraction (chloroform extractable C-subunit). (a) Western blot using C-subunit-specific antibodies shows that mPTP activation in isolated mitochondria (+Ca²⁺) increases the amount of C-subunit associated with channel-forming 'PTP pore' fraction. Note that the amount of complex is dramatically decreased if mPTP is inhibited with 5 μM Ruthenium Red (Rut Red) or 1 μM CSA despite the presence of the same amounts of Ca²⁺ (200 μM). The samples for analysis were purified using chloroform extraction protocol as described in Pavlov et al.¹² and identical to the samples used for mPTP monitoring in Figure 1. (b) Densitometry analysis of western blots (as in a) from three independent experiments (*P<0.01 t-test). (c) Western blot of C-subunit immunoprecipitated from the total mitochondrial lysate using C-subunit antibodies shows similar total amounts of C-subunit in different samples. Note high molecular bands presumably formed by oligomers of the C-subunit.