Anti nrf1

To assess protein level in brain or cells, lysates were prepared in SDS lysis buffer containing protease inhibitors and phosphatase inhibitors. The amount of proteins in lysates were normalized by BCA protein assay kit (Millipore). Proteins were resolved by SDS–polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes (PerkinElmer). Transferred blots were incubated with primary antibodies, anti-PGAM5 (1:500, Santa Cruz, SC-515880), anti-GAPDH (1:5000, Genetex, GTX100118), anti-TUJ1 (1:5000, BioLegend, #801202), anti-GFAP (1:5000, Genetex, GTX108711), anti-PGC1α (1:500, Genetex, GTX37356), anti-NRF1 (1:1000, Genetex, GTX103179), anti-TFAM (1:500, Genetex, GTX59889), or anti-TIM23 (1:500, Santa Cruz, SC-514463) overnight. Then the blots were incubated with secondary antibodies, IRDye 800CW goat anti-rabbit IgG secondary antibody (1:1000, LI-COR, # 926-32211) or goat anti-mouse IgG secondary antibody Alexa Fluor™ 700 (1:1000, Invitrogen, # A-21036), for 1 h. Membranes were imaged by ChemiDoc™ MP Imaging System (Bio-Rad) and the signal intensity of bands was quantified by Image Lab software (Bio-Rad, version 6.1.0) (Additional file 2).

The primary materials used in the present study were as follows: fetal bovine serum (FBS), bupivacaine, lipid emulsions, puerarin injections, pentobarbital sodium, the RNeasy Mini Kit (QIAGEN), the QuantiNova SYBR Green PCR Kit (QIAGEN), the QuantiNova Reverse Transcription Kit (QIAGEN), Anti-UCP2 (GeneTex), Anti-NRF1 (GeneTex), Anti-SLC25A6 (GeneTex), Anti-mtTFA (GeneTex), Anti-VDAC1, Anti-PGC-1 (Abcam), Anti-Bcl-2 (Abcam) and ATP assay kit (Abcam, ab83355). JC-1 kit (Beyotime, C2006), Fluo-3 AM (Abcam), DCFH-DA (Solarbio), Rat COX ELISA kit (CUSABIO), MDA ELISA Kit (Elabscience).