Enhanced chemi luminescence (ecl)

Cell Radioimmunoprecipitation assay buffer (RIPA) lysis buffer (P0013B, Beyotime, Shanghai) containing phenylmethanesulfonylfluoride (PMSF) was used to lyse the cells. Afterward, 20–40 g of proteins were electrophoresed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS/PAGE), which were transferred onto the polyvinylidene fluoride (PVDF) membranes subsequently. The corresponding primary antibodies were taken to incubate with membranes overnight after blocking in nonfat milk for 2 h. Followed by a second antibody (1:3000, Biosharp, China) incubation at room temperature for an hour. The primary antibodies were listed as follows: anti-TM7SF2 (1:1000), anti-CPT1A (1:1000), anti-GAPDH (1:3000), anti-Tubulin (1:3000), anti-Flag (0.8 mg/mL), anti-HA (0.8 mg/mL), anti-WNT3A (1:1000), anti-β-catenin (1:1000), anti-c-Myc (1:1000) and anti-TCF1 (1:1000). Protein bands were observed by adding enhanced chemiluminescence (ECL, meilunbio, China) reagent on the bands. Finally, ImageJ software was used to calculate relative protein expression.

The collected cell and liver tissue was treated by cell lysis solution. And then, we used BCA protein assay kit (Thermo, America) to detect the concentration. With the help of 10% SDS‐polyacrylamide gel, target substance was obtained and it was transferred to polyvinylidene fluoride membrane. After blocked, incubated by particular primary antibody and secondary antibody, we analysed the bolt with Image‐Pro Plus, and we detected the signal with the assistance of ECL (Dalian Meilun Biotechnology Co., Ltd).

Cells were seeded into a 15 cm dish at 4.5 × 10⁶ cells/well and treated with Nuplazid for 24 h. Total protein was extracted from cells using RIPA lysate buffer. Protein samples (30 μg, quantified by the BCA Protein Assay Kit) were boiled with loading buffer at 98 °C for 8 min and then were subjected to SDS–PAGE and transferred to a PVDF membrane (Immobilon®-P Membrane). After blocking with 5% non-fat milk for 1 h at room temperature, the membrane was incubated with primary antibody at 4 °C overnight. The next day, the membrane was incubated with the corresponding secondary antibody at room temperature for 2 h after being washed three times with TBST. Protein bands were visualised using the enhanced chemiluminescence (ECL, Meilunbio®).

Total cell proteins were extracted using a 99:1 mixture of cell lysate and protease inhibitor, added to loading buffer and then boiled in boiling water for 5 min. Denatured protein samples were electrophoresed on 10% SDS/polyacrylamide gel (SDS/PAGE), and then the proteins on the gel were transferred to PVDF membrane. The PVDF membranes were closed with 5% milk powder and incubated with primary antibodies for N-cadherin (United Kingdom, Abcam,ab125219,1:500), vimentin (United Kingdom, Abcam, ab125219, 1:500) and β-actin (China, Bioss, AH11286402), followed by incubation with secondary antibody (China, absin, abs20040ss, 1:5,000). Finally, exposure development was performed using an enhanced chemiluminescence kit (ECL; China, Meilunbio, MA0186).

NP-40 lysis buffer supplemented with protease inhibitors (Meilun, China) was used to extract total protein. BCA Reagent (Thermo Scientific, Rockford, IL, USA) was used to measure the protein concentration. We used an SDS–polyacrylamide gel was to separate the total proteins and transferred proteins to 0.45 μm PVDF membrane (Millipore, USA). The primary antibodies used for western blot analysis are listed as follows: YAP (Proteintech, 66,900–1-Ig), ATXN3 (Proteintech, 13,505–1-AP), GAPDH (Proteintech, 60,004–1-Ig), Myc (Proteintech, 60,003–2-Ig), HA (Proteintech, 51,064–2-AP) antibodies. Signals were detected and visualized using ECL (Meilun, China) and ChemiDocMP imager (Bio-Rad).

Total proteins that were extracted from EL4 cells with RIPA buffer containing phosphatase inhibitor as well as protease inhibitor were separated in 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and were subsequently transferred to polyvinylidene fluoride (PVDF) membranes (Merck Millipore). After PVDF membrane was blocked with 5% nonfat milk for 2 h, it was sequentially incubated overnight at 4 ℃ with the following primary antibodies: PPARγ (CST, Danvers, MA, USA), ELK1 (CST), ETS1 (Proteintech, Rosemont, USA), Foxp3 (Abcam, Cambridge, UK), and Sp1 (Abcam). Horseradish peroxidase (HRP)-labeled goat anti-mouse antibody (Proteintech) or HRP-labeled goat anti-rabbit antibody (Proteintech) was utilized as the secondary antibody [22 (link)]. The proteins were subsequently revealed with enhanced chemiluminescence (ECL, Meilunbio, Dalian, China). The density of the bands (versus GAPDH) was analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Cells (4.5 × 10⁶) were plated on a 15 cm dish and treated with various concentrations of vortioxetine hydrobromide (0, 0.5, 1, 2 and 4 μM) for 24 h. Then the cells were collected and lysed with RIPA buffer. The protein concentration was detected using the BCA kit (BCA Protein Assay Kit, Beyotime Biotechnology, Shanghai, China). Protein samples were heated with protein loading buffer at 100 °C for 5 min and then were separated by SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked with 5% nonfat milk for 1.5 h, incubated with primary antibodies overnight at 4 °C. Next, the membranes were incubated with corresponding secondary antibodies for 1.5 h and were detected using a chemiluminescence reagent (ECL, Meilunbio).