Supersignal west femto maximum sensitivity substrate detection system

Total cells were lysed with RIPA lysis buffer with protease inhibitor and phosphatase inhibitor cocktail (complete cocktail; Roche Applied Science). Equal amounts of the sample proteins were electrophoresed on sodium dodecyl sulphate polyacrylamide gel before being transferred to a polyvinylidene fluoride membrane with 0.45 μm pores (Millipore, Billerica, MA). Membrane was then incubated with first antibodies followed by second antibodies. Antibodies used are listed in Table S2, Supporting Information, and immunoblotting with anti‐β‐actin antibody was conducted to ensure equal protein loading. Signals were measured using Super Signal West Femto Maximum Sensitivity Substrate detection system (Thermo Scientific, Waltham, MA). Images of uncropped blots are shown in Figure S11A–N, Supporting Information.

Western blotting was performed as previously described [64 (link)]. For cell culture experiments, cells were collected and lysed with RIPA lysis buffer with protease inhibitor and phosphatase inhibitor cocktail (complete cocktail; Roche Applied Science, Mannheim, Germany). For mouse experiments, the gastrocnemius muscle was isolated and immediately homogenized with RIPA lysis buffer with protease inhibitor and phosphatase inhibitor cocktail (complete cocktail; Roche Applied Science) to obtain protein extracts. Equal amounts of the sample proteins were electrophoresed on sodium dodecyl sulfate polyacrylamide gel and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA). The antibodies used are listed in Supplementary Table 3, and the signal was measured by the SuperSignal West Femto Maximum Sensitivity Substrate detection system (Thermo Scientific, Waltham, MA). β-actin was used as a loading control. The quantitative analysis was performed using Quantity One (Thermo Scientific), and the results were shown as relative to the expression level in the corresponding controls, which are assumed as 1.

Cells were lysed in RIPA (Sigma) lysis buffer containing 1x Halt™ Protease and Phosphatase Inhibitor Cocktail (ThermoFisher). Protein concentration was measured by Bio-Rad Bradford reagent. Protein samples were prepared by addition of 4x Laemmli Sample buffer (Bio-Rad) and 2-mercaptoethanol (Bio-Rad) and resolved on 4–12% SDS-PAGE (Sodium dodecyl sulfate–polyacrylamide) gels, which were subsequently transferred to PVDF membranes (Bio-Rad) using Trans-Blot Turbo transfer buffer (Bio-Rad) and a Trans-Blot Turbo Transfer System (Bio-Rad). Membranes were blocked for 1 h at room temperature with 5% blotting-grade blocker non-fat dry milk (Bio-Rad), followed by overnight 4 °C incubation with the appropriate primary antibody and 1 h room temperature incubation with an anti-rabbit or anti-mouse IgG (H + L)-HRP conjugate (Bio-Rad) secondary antibody. Blots were imaged using Supersignal West Femto Maximum Sensitivity Substrate detection system (Thermo) and the ChemiDoc Imaging System (Bio-Rad). The following primary antibodies were used: c-Myc (Y69) (Abcam #ab32072, 1:1000 dilution), NME2 (4G7A8) (Abcam #ab204958, 1:1000 dilution), GAPDH (Cell Signaling Technology #3683, 1:5000 dilution), Actin (Cell Signaling Technology #5125 S, 1:5000 dilution).

Cells were prepared as described above before being lysed using RIPA lysis buffer with protease inhibitor and phosphatase inhibitor cocktail (complete cocktail; Roche Applied Science). For clinical tissues and xenografted tumors, proteins were extracted from the frozen specimens using RIPA lysis buffer with protease inhibitor and phosphatase inhibitor cocktail. Equal amounts of proteins (20 μg) were electrophoresed on sodium dodecyl sulfate polyacrylamide gel and transferred to a polyvinylidene fluoride (PVDF) membrane with 0.45 μm pores (Millipore, Billerica, MA). The signals were measured using SuperSignal West Femto Maximum Sensitivity Substrate detection system (Thermo Scientific, Waltham, MA). Antibodies used were listed in S2 Table. β-actin was used to normalize sample amplifications.

Total cells were lysed with RIPA lysis buffer with protease inhibitor and phosphatase inhibitor cocktail (complete cocktail; Roche Applied Science, Mannheim, Germany). Equal amounts of the sample proteins were electrophoresed on sodium dodecyl sulphate polyacrylamide gel before being transferred to a polyvinylidene fluoride membrane with 0.45 µm pores (Millipore, Billerica, MA). Membranes were then incubated with first antibodies followed by second antibodies. Antibodies used are listed in Table S2 (Supporting Information), and immunoblotting with anti‐β‐actin antibody was conducted to ensure equal protein loading. Signals were measured using Super Signal West Femto Maximum Sensitivity Substrate detection system (Thermo Fisher Scientific, Waltham, MA). For xenografted tissues and clinical HCC samples, tissues were frozen in liquid nitrogen and ground before being lysed with RIPA lysis buffer with protease inhibitor and phosphatase inhibitor cocktail (complete cocktail; Roche Applied Science). Western blotting was performed as described above, and immunoblotting with anti‐GAPDH antibody was conducted to ensure equal protein loading for samples from xenografted tissues. Images of uncropped blots are shown in Figure S17 (Supporting Information) (A to M).