Rabbit anti hk2

The cells were washed three times with PBS, and protein lysate buffer containing 5 µL PMSF (1 mM) per 500 µL was placed on ice for 5 min and centrifuged at 14,000×g for 10 min after sonication. Then, the supernatant was collected. The protein concentration was measured using a bicinchoninic acid (BCA) protein assay kit (Beyotime, Shanghai, China). After boiling with loading buffer for 10 min, equal amounts of protein (40 µg) were separated by 10% SDS–PAGE (Beyotime, Shanghai, China) and transferred onto PVDF membranes (Millipore, Massachusetts, USA). Membranes were blocked with 5% milk powder for 60 min at room temperature and incubated with specific primary antibodies at 4 °C overnight. Rabbit anti-SIRT6 (1:1000 dilution, Abcam, UK), rabbit anti-HIF-1α (1:500 dilution, Abcam, UK), rabbit anti-HK2 (1:500 dilution, Cell Signaling Technology, USA), and mouse anti-β-actin (1:1000 dilution, ZSGB-Bio, Beijing, China) antibodies were used. Proteins were detected using anti-rabbit IgG (1:2000 dilution, Cell Signaling Technology, USA) or anti-mouse IgG (1:2000 dilution, Cell Signaling Technology, USA) antibodies and a chemiluminescence detection system (FluorChem HD2, USA).

Cells were fixed in cold 4% paraformaldehyde in PBS for 10 min. NPCs and neurons were permeabilized at room temperature for 15 min in 0.2% TritonX-100 in PBS. Samples were blocked in 5% BSA with 0.1% Tween 20 for 30 min at room temperature. The following primary antibodies and dilutions were used: goat anti-Sox2 (Santa Cruz Biotechnology, Dallas, TX), 1:200; mouse anti-Nestin (EMD Millipore, Temecula, California), 1:200; rabbit anti-βIII-tubulin (Covance, San Diego, CA), 1:200; mouse anti-βIII-tubulin (Covance), 1:200; rabbit anti-GFAP (Dako, Carpinteria, CA) 1:200; mouse anti-MAP2AB (Sigma-Aldrich, St. Louis, MO), 1:200; rabbit anti-LDHA (Cell signaling, Danvers, MA), 1:200, and rabbit anti-HK2 (Cell signaling), 1:200. Secondary antibodies were Alexa donkey 488 and 568 anti-mouse, rabbit and goat (Invitrogen, Carlsbad, CA), used at 1:1000. Nuclear staining was done with Hoechst (Invitrogen).