To verify the effect of formononetin on the mPTP opening, we examined the formation of protein complex between two main mPTP components ANT and Cyp-D. Following immunoprecipitation with anti-ANT antibody, we analyzed the presence of ANT and Cyp-D by Western blotting with specific antibodies.
The mPTP opening may be regulated by posttranslocational modification of Cyp-D and physical interaction of Cyp-D with matrix proteins (e.g., p53, PPAR alpha) .
Upon activation, Akt and PKC subsequently phosphorylate GSK-3[beta] at Ser9, leading to direct inhibition of GSK-3[beta] activity towards the mPTP opening and the disruption of the protein complex involving ANT and Cyp-D. Ultimately, formononetin attenuated the mPTP opening and maintained mitochondrial membrane integrity.
The pull-down materials were analyzed by immunoblotting (IB) with antibodies against ANT and Cyp-D. (b) Immunoprecipitation with antibody against phospho-GSK-3[beta] (Ser9).