The detailed protocol was elaborated previously.(13) For ubiquitination assay, cells were co-transfected with indicated plasmids for 24 h. Subsequently, cells were incubated with MG132 (20 μM) for 4 h to prevent proteasome degradation. Cell lysates were immunoprecipitated and analyzed using immunoblotting with indicated antibodies. The antibodies are listed as follows: UQCRC2, SDHB, NDUFB8, MTCO1, ATP5A, ALKBH5, TRIM56,TFAM, p65, p-p65, DRP1, BAX, AMPKα1/α2 (Abcam, USA), p-DRP1 (Ser616) (Cell signaling, USA), AMPKα, PGC-1α, Myc-tag, Flag-tag, HRP-conjugated goat anti-rabbit/mouse IgG, and β-actin (Proteintech, USA).
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