Western blotting, immunoprecipitation and ubiquitination assay

ZL Zhimei Lv
ZW Ziyang Wang
JH Jinxiu Hu
HS Hong Su
BL Bing Liu
YL Yating Lang
QY Qun Yu
YL Yue Liu
XF Xiaoting Fan
MY Meilin Yang
NS Ning Shen
DZ Dongdong Zhang
XZ Xia Zhang
RW Rong Wang
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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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