Anti hk2 2867s

Human astrocytes, NHA, which is a normal human cell line derived from human brain tissue, and two neuroblast cell lines, IMR-32 and SK-N-SH were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Invitrogen, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin at 37 °C under 5% CO₂ supply. The short hairpin RNA (shRNA) against ACAP2 (sh ACAP2), miR-143-3p mimics, and overexpression plasmids of ACAP2 (pcDNA3,1) and HK2 (pcDNA3.1) as well as their negative controls were obtained from GenePharma (Shanghai, China). The shRNA sequences were: shcirc_ACAP2: Forward: 5'-GGCAGCATACAGGAAGATGAA-3', Reverse: 5'-UUCAUCUUCCUGUAUGCUGCC-3'; Negative control: Forward: 5'-UUCUCCGAACGUGUCACGUUUC-3', Reverse: 5'-GAAACGUGACACGUUCGGAGAA-3'. Cells were seeded onto 6-well plates and cultured to 60–70% confluence, then transfected with shRNA, overexpression plasmid, or miRNAs using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Transfections were conducted for 48 h, followed by cell collections for downstream experiments. Rabbit anti-β-actin (#4970) and anti-HK2 (#2867S) were purchased from Cell Signaling Technology (CST; Danvers, MA, USA).

Western blotting assays were carried out using standard procedures. The antibodies included anti-HK2 (2867S, Cell Signaling Technology), anti-FSP1 (#13018, Cell Signaling Technology), anti-GLUT1 (12939S, Cell Signaling Technology), and anti-FAP (ab53066, Abcam). Antibodies for horseradish peroxidase-conjugated goat anti-rabbit/anti-mouse IgG and β-actin were purchased from Sigma (St. Louis, MO, USA).