Verification of O-GlcNAcylated mitochondrial proteins.

<p>First, three O-GlcNAcylated mitochondrial proteins, cytochrome P450 (CP1A1+1A2), ATP synthase subunit beta (ATPB) and Long-chain-fatty-acid--CoA ligase 1 (ACSL1), were immunoprecipitated from rat liver mitochondrial proteins respectively. Then, the purified proteins were analyzed by silver staining and western blot with the specific antibodies. Stringently, goat anti-rat IgG-CFL 594 was also used to exclude non-specificity. Furthermore, the proteins were detected with RL2 and CTD110.6 to confirm their O-GlcNAcylation. Strictly, the competition experiments with 1 M GlcNAc was performed to exclude nonspecific binding of RL2 and CTD110.6 antibody.