Polyvinylidene fluoride pvdf membrane

Western blot was used to quantitatively determine the expression of ED-B FN in tumor tissues and normal liver tissues. Firstly, tissues were lysed in RIPA buffer (Beyotime Biotechnology, Shanghai, China) supplemented with PMSF (Beyotime Biotechnology, Shanghai, China). Protein concentration was determined by the BCA protein assay kit. Equal protein from each sample was mixed with 5× sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample loading buffer (Beyotime Biotechnology, Shanghai, China) and separated by SDS-PAGE on 8% (v/v) resolving gel. The protein was transferred to the Polyvinylidene Fluoride (PVDF) membrane (Beyotime Biotechnology, Shanghai, China). Membranes were blocked with fast blocking and incubated with anti-EDB-FN (G4, Absolute Antibody, Oxford, UK) and anti-β-actin (Beyotime Biotechnology, Shanghai, China) primary antibodies overnight. Membranes were washed and incubated with horseradish peroxidase-conjugated anti-rabbit IgG secondary antibodies (Beyotime Biotechnology, Shanghai, China) for 45 minutes. Membranes were activated with Immobilon Western Chemiluminescent HPR Substrate and imaged with the tanon 5200 chemiluminescent imaging system.

The CD34+CD38− LSCs cells were lysed using protein lysis buffer (Applygen Tech. Inc., Beijing, China). The protein concentration of the lysates was evaluated using the bicinchoninic acid (BCA) protein concentration detection kit (Cat. No. P0012) (Beyotime Biotech., Shanghai, China). The lysates were separated with 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Sigma-Aldrich, St. Louis, MO, USA) and transferred onto polyvinylidene fluoride (PVDF) membranes (Beyotime Biotech., Shanghai, China). The PVDF membranes were incubated overnight at 4°C in the primary antibodies, which were all purchased from Abcam Biotech (Cambridge, MA, USA). The primary antibodies included rabbit anti-human SIRT1 monoclonal antibody (Cat. No. ab32441) (1: 2000), rabbit anti-human TSC2 monoclonal antibody (Cat. No: ab190349) (1: 2000), rabbit anti-human polyclonal antibody (Cat. No. ab8227) (1: 2000). The membranes then incubated in the secondary horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Cat. No. ab6721) (1: 1000) (Abcam Biotech., Cambridge, MA, USA) at 37°C for 1 h. Western blot images were visualized using the electrochemiluminescence (ECL) kit (Pierce Thermo Scientific, Rockford, IL, USA).

Whole cell protein lysates were obtained using RIPA buffer (cat. no. P0013E; Beyotime) supplemented with 1% phenylmethanesulfonyl fluoride (PMSF; cat. no. ST506; Beyotime), and protein concentrations were quantified using the BCA Protein Assay Kit (cat. no. P0012; Beyotime). Proteins (40 µg) were separated on SDS-PAGE gels (cat. no. P0012A; Beyotime) and then transferred onto polyvinylidene fluoride (PVDF) membranes (cat. no. IPVH00010; Millipore). Membranes were blocked with 5% nonfat milk and then incubated with the following primary antibodies: CCNB1 (cat. no. #12231; RRID: AB_2783553) and CDK1 (cat. no. #28439; RRID: AB_2798959) (purchased from Cell Signaling Technology); cyclin-dependent kinase inhibitor 1 (CDKN1A; cat. no. 10355-1-AP; RRID: AB_2077682) and cadherin-1 (CDH1; cat. no. 20874-1-AP; RRID: AB_10697811) (purchased from Proteintech Group Inc.); and cadherin-2 (CDH2; cat. no. ab98952; RRID: AB_10696943), snail family transcriptional repressor 2 (SNAI2; cat. no. ab27568; RRID: AB_777968) and twist family bHLH transcription factor 1 (TWIST1; cat. no. ab50581; RRID: AB_883292) (purchased from Abcam); all diluted 1:1,000. After incubation with secondary antibodies, a chemiluminescent horseradish peroxidase (HRP) substrate (cat. no. P90719; Millipore) was added, and the blots were imaged.