Pkm2 d78a4 xp rabbit mab

Wild-type (C57BL/6 J) (n = 21) and L.G6pc^−/− male mice (n = 60) were fed a high fat/high sucrose (HF/HS) diet throughout the course of the study (and 3 months prior to beginning treatment with mRNAs) to accelerate/facilitate the development of HCA/HCCs^{54 (link)}. Male mice were treated with 8–10 consecutive injections of either PBS, eGFP mRNA, or hG6PC-S298C mRNA administered i.v. every 7 to 14 days at 0.25–0.5 mg/kg dose level. Mice were euthanized 8 days after the last mRNA treatment and livers and tumors were harvested, weighed, counted, measured, and photographed. Liver and tumor tissues were either, snap-frozen in liquid nitrogen and kept at −80°C, or fixed and embedded in paraffin blocks for further use. Total RNA was extracted from liver tissue by the Promega Maxwell RSC simplyRNA tissue kit as mentioned above, and the HCA/HCC-related mRNA markers (β-catenin, transforming growth factor beta-1 and glutamine synthetase) were measured by custom Taqman assays from ThermoFisher Scientific (Supplementrary Table 3). HCA/HCC-related protein markers (PKM2, β-catenin, and p62 were measured by standard immunoblotting procedure as described above with the following primary antibodies: PKM2 (D78A4) XP^® Rabbit mAb (cat #4953, Cell Signaling), β-catenin (D10A8) XP^® Rabbit mAb (cat #8480, Cell signaling), and Anti-SQSTM1/p62 mouse mAb (cat #ab56416, Abcam).

Proteins were extracted by a total protein extraction kit and quantified by a BCA protein assay kit (CoWin Biotech, Beijing, China). About 20 μg of proteins were separated in 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to polyvinylidine difluoride membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5% skimmed milk powder in TBST buffer and then incubated with primary antibodies (anti-Hexokinase II antibody, 1:1000 dilution, Abcam; anti-Fructose 6 Phosphate Kinase antibody, 1:1000 dilution, Abcam; PKM2 (D78A4)XP® Rabbit mAb, 1:1000 dilution, Cell Signaling Technology; LDHA (C4B5) Rabbit mAb, 1:1000 dilution, Cell Signaling Technology; anti-Glucose Transporter GLUT1 antibody, 1:5000 dilution, Abcam; β-actin Rabbit mAb, 1:1000 dilution, Cell Signaling Technology) at 4°C overnight. After being washed, membranes were incubated with corresponding horseradish peroxidase-conjugated secondary antibodies (anti-rabbit IgG or anti-mouse IgG, HRP-linked antibody, Cell Signaling Technology, Danvers, MA, USA), and signals were visualized by the enhanced chemoluminescence method (Millipore, Bedford, MA, USA). The relative expression levels of proteins were quantified by ImageJ software with β-actin as the loading control.