Anti pgk1

Porcine kidney-15 (PK-15) and human embryonic kidney (HEK293T) cells were cultured at 37°C and 5% CO₂ in Dulbecco’s modified Eagle’s medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco), 100 IU/mL penicillin, 100 g/mL streptomycin, and 2 mM L-glutamine (Gibco). The SVA strain, CH-GDFS-2018, was isolated by our group and propagated and titrated in PK-15 cells using the Reed–Muench method [46 (link)]. The SVA strain was incubated in a water bath at 70°C for 2 h to generate a heat-inactivated SVA (Heat-SVA).
Sodium oxamate, 2-deoxyglucose (2DG), sodium lactate, dichloroacetate (DCA), and glucose were purchased from Macklin (Shanghai, China). The 2DG (50 mM), sodium oxamate (25 mM), DCA (5 mM), sodium lactate, and glucose were directly solubilized in DMEM or phosphate buffered saline (PBS) at the indicated concentrations. DMEM, PBS, and FBS were obtained from Gibco (Grand Island, NY, USA). Anti-HIF-1α, Anti-PGK1, anti-PKM, anti-GAPDH, anti-RIG-I and anti-β-actin primary antibodies were purchased from Santa Cruz (Shanghai, China). A primary antibody of rabbit anti-VP2 of SVA was prepared and stored in our laboratory [27 (link)]. The respective horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from Abcam (Cambridge, UK).

The cells were washed twice with PBS,
collected by scraping in radioimmunoprecipitation assay buffer with
EDTA-free complete protease inhibitors (Roche), and sonicated on ice
for 15 s (1 s on/off cycles). Insoluble debris was cleared by centrifugation
at 16,000g and 4 °C for 15 min. The supernatant
was diluted into 4× Laemmli buffer containing 100 mM βME,
heated to 95 °C for 5 min, cooled to room temperature, resolved
on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis
gel, and transferred onto a nitrocellulose membrane by standard Western
blotting methods. The membranes were blocked in 2% BSA in TBS + 0.1%
Tween-20 (TBST). The antibodies used in this study include: anti-MRP1
(1:1000, #72202, Cell Signaling Technology) and anti-PGK1 (1:3000,
sc-130335, Santa Cruz Biotechnology). Secondary donkey antirabbit
680 and donkey antimouse 800 (1:10,000, LiCor). The blots were imaged
on a LiCor infrared scanner and images processed in ImageJ.

Western blotting was conducted as described previously14 (link)17 (link)23 (link) with some modifications. Briefly, transiently transfected MONOMAC-6 cells were collected and lysed with RIPA buffer (Thermo Scientific, BufferRockford, IL). Proteins from the lysate were fractionated by electrophoresis through 4–15% polyacrylamide gels (Bio-rad, Hercules, CA) and transferred to polyvinylidene fluoride membranes using tris-glycine transfer buffer (Thermo Scientific). Blots were incubated with IRDye 800CW-conjugated or 700CW-conjugated antibody and infrared fluorescence images were obtained with the Odyssey infrared imaging system (Li-Cor Bioscience, Lincoln, NE). 100-200 ng ml⁻¹ anti-CRTC1, anti-FLT3, anti-MYCBP, anti-BMI1, anti-CDK6, anti-PGK1 (Santa Cruz Biotechnology Inc.), anti-HMGA1 (Abcam) and anti-GAPDH (Thermo Scientific) antibodies were used to detect corresponding proteins. Original scans can be found as Supplementary Figures 7–9.