Sc 492

Six rats in each group at different time points had brain tissues harvested for western blot analysis. Western blot was performed as previously described (Xu et al., 2017 (link)). Briefly, frozen perihematoma tissues (basal ganglia) were homogenized in RIPA lysis buffer (Beyotime, Shanghai, China). Then the protein samples were separated by 10% or 12% SDS-PAGE, and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore). Then, the PVDF membranes were blocked with 5% bovine serum albumin for 1 h and incubated with the primary antibodies overnight, including: anti-sirt3 antibody (1:500, Abcam, ab86671), anti-NRF1 antibody (1:2000, Abcam, ab175932), anti-TFAM (1:1000, Abcam, ab131607), anti-SOD2 (1:5000, Abcam, ab13533), anti-Ac-SOD2 (1:1000, Abcam, ab137037), anti-cleaved caspase-3 (1:1000, CST, cst#9661), anti-Bax (1:1000, CST, cst#2772), anti-Bcl-2 (1:800, SantaCruz, sc-492), anti-NLRP3 (1:1000, ab210491, Abcam), anti-interleukin (IL)-1β (1:2000, Santa Cruz, sc-23459), and β-actin (1:5000, Abcam, ab8226). Then, the PVDF membranes were disposed with relevant secondary antibodies (1:5000) for 1 h at normal temperature. The signals of protein bands were detected with ChemiDoc detection system and quantified using Quantity One software (Bio-Rad, Hercules, CA, United States).

The SN and striatum were prepared in RIPA buffer containing phosphate and protease inhibitor cocktails. After 30 min in an ice-bath, tissue lysate was obtained by centrifugation at 12,000 rpm for 10 min. The tissue lysate was mixed in a loading buffer and boiled for 5 min. Then, equivalent amounts of protein were separated on a 10% SDS-polyacrylamide gel and transferred onto PVDF membranes. Membranes were blocked with 5% BSA in Tris-buffered saline. Anti-TH (1:300, Abcam, ab112), anti-Bcl2 (1:100, Santa, Sc492), anti-Bax (1:500, Abcam, ab32503), Cyt C (1:500, Abcam, ab13575), and cleaved-caspase 3 (1:500, CST, 9662) were diluted in 1% BSA and incubated overnight at 4°C, followed by incubation for 2 h at room temperature with anti-rabbit IgG HRP (1:5000, Abcam, ab6721) or anti-mouse IgG HRP (1:5000, Abcam, ab6789). Immunoblotting was stripped and reprobed with antibodies to GAPDH (ab181602, 1:3000) as an internal control. Blots were quantified by an Epson V330 Photo Scanner (Seiko Epson Co., Nagano, Japan) and densitometry was performed with Quantity One v.4.6.2.