Atp5a1

The cerebral ischemic cortex was homogenized in RIPA buffer. The homogenized protein was centrifuged at high speed, then the supernatant was extracted and the precipitation was discarded. Then the total protein concentration was determined with the BCA kit. 20 μg total protein samples from cortex were separated using SDS-PAGE and transferred to a PVDF membrane. The membranes were blocked using blocking solution (5% non-fat milk in TBST, pH 7.4) at room temperature for 60 min. Discard blocking solution and incubation with the primary antibody overnight at 4°C. Primary antibodies included: Opa1 (1:2000, Proteintech), Mfn1 (1: 2000, Proteintech), Mfn2 (1: 2000, Proteintech) and β-actin (1: 5000, Proteintech), AMPK (1:4000, Abclonal), pAMPK (1:5000, Abclonal), NDUFB8 (1:500, Abclonal), SDHB (1:500, Abclonal), UQCRC2 (1:500, Abclonal), MTCO2 (1:500, Abclonal), ATP5A1 (1:500, Abclonal). The following day, membranes were washed with TBST (10 min × 3) each time and subsequently incubated with secondary antibodies (1: 8000) for 2 h at room temperature. Membranes were then washed three times with TBST (10 min × 3). Protein expression was examined by using Chemiluminescent HRP Substrate (Millipore) and Cytiva. Densitometric values was quantified with ImageJ and were normalized with respect to β-actin immunoreactivity to correct for any loading and transfer differences among samples.

Immunostaining was performed as previously described (Gao et al., 2017 (link)). Briefly, cells for immunostaining were seeded onto Matrigel pretreated coverslips. After aspiration of medium, cells were fixed with 4% PFA for 15 min at room temperature. Then cells were treated with permeabilization and blocking buffer (1% BSA and 0.5% Triton-X 100 in PBS) for 1 h at room temperature. Primary antibodies were incubated overnight at 4°C. The next day, fluorescent-dye conjugated secondary antibodies were incubated for 1 h at room temperature. Slides were mounted and images were captured with Leica SP8 microscope. The following antibodies were used in this study: TOM20 (Santa Cruz, #sc-17764), ATP5A1 (Abclonal, #5884), LAMP1 (Abcam, #24170), OCT4 (Santa Cruz, #sc-9081), NANOG (Santa Cruz, #sc-33760), SOX2 (Santa Cruz, #sc-17320), S100B (Sigma, #S2532), GFAP (Dako, #Z033401; Thermo Fisher, #13-0300), CRYAB (Santa Cruz, #sc-137143), TUJ1 (Biolegend, #801202), DCX (Santa Cruz, #sc-8066), MAP2 (Millipore, #AB5622), NEUN (Millipore, #ABN78), and SYN1 (Millipore, #Ab1543).

The cell and tissue lysates were extracted using RIPA lysis buffer containing protease and phosphatase inhibitor cocktail (NCM Biotech, Suzhou, China). Equivalent amounts of protein were electrophoresed on SDS-PAGE gels followed by transferring to PVDF membranes (Millipore, Billeria, MA). Antibodies against SNAP23 (Proteintech, China), TSG101, CD9 (Abcam, USA), Calnexin, Lin28a, Cyclin-D1, c-Myc, PKM2, p-PKM2 (Tyr105) (Cell Signaling Technology, USA) and ATP5A1, SDHA, CYTb, COX1 (ABclonal, Wuhan, China) were used for immunoblotted. β-Actin (Proteintech, China) served as the loading control. Protein bands were visualized and analyzed using the Odyssey Imaging System (LI-COR, USA) and Image J software.