Total protein extraction was conducted as previously reported [15 (link)]. A mitochondrial isolation kit was purchased for (GENMED, China) mitochondrial protein isolation. Twenty micrograms of total protein or mitochondrial protein from cells, mice tissues and human samples were resolved and then transferred to PVDF membranes. Afterwards, 5% blotting-grade blocker (Bio-rad, USA) or BSA albumin fraction V (Biofroxx, USA) was provided for 90 min to block the PVDF membrane. Primary antibodies for immunoblotting against SIRT1 (1:10000, ab189494, Abcam), Akt (1:1000, 9272S, Cell Signaling), p-Akt (1:1000, 9271S, Cell Signaling), PDK1 (1:1000, 5662S, Cell Signaling), p-PDK1 (1:1000, AF3018, Affinity), cytochrome C (1:1000, ab133504, abcam), Bcl2 (1:1000, 12,789–1-AP, Proteintech), BAX (1:1000, ab32503, abcam), VDAC (1:10000, CY5416, Abways) and β-actin (1:10000, ap0060, Bioworld Technology), the goat anti-rabbit HRP-conjugated secondary antibody was then incubated (1:10000, a0045, sigma). The antibodies used in our study have been previously validated in former works [15 (link), 16 (link)]. Diaminobenzidine was used to detect protein signals and ImageJ software (NIH, Bethesda, MD, USA) was used to perform measurement of protein expression in a blind fashion.
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