A549 and H446 cells were treated with shikonin [0 (control), 10, 20 or 50 µM] for 24 h at 37°C, then total protein was extracted from cells using RIPA lysis buffer (Santa Cruz Biotechnology, Inc.) supplemented with protease and phosphatase inhibitor mixture. Total protein was quantified using a BCA assay and 50 µg protein/lane was separated via 10% SDS-PAGE. The separated proteins were subsequently transferred onto PVDF membranes (EMD Millipore) and blocked with 5% skim milk for 2 h at room temperature. The membranes were then incubated with the following primary antibodies at 4°C overnight: Anti-PFKFB2 (1:1,000; cat. no. ab234865; Abcam), anti-PDK1 (1:2,000; cat. no. ab202468; Abcam), anti-glucose transporter 1 (GLUT1; 1:200; cat. no. ab150299; Abcam), anti-phosphoglycerate kinase 2 (PGK2; 1:1,000; cat. no. ab183031; Abcam), anti-lactate dehydrogenase A (LDHA; 1:1,000; cat. no. ab101562; Abcam), anti-PKM2 (1:1,000; cat. no. ab137852; Abcam), anti-GLUT3 (1:8,000; cat. no. ab41525; Abcam), anti-pyruvate dehydrogenase (PDH; 1:1,000; cat. no. 3205; Cell Signaling Technology, Inc.), phosphorylated (p)-PDH (1:1,000; cat. no. 31866; Cell Signaling Technology, Inc.) and anti-GAPDH (1:2,500; cat. no. ab9485; Abcam). Following the primary antibody incubation, the membranes were incubated with an anti-rabbit HRP-conjugated secondary antibody (1:5,000; cat. no. sc-2357; Santa Cruz Biotechnology, Inc.) for 1 h at room temperature. Protein bands were visualized using an enhanced chemiluminescence kit (EMD Millipore).

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