Ecl kit

The samples were lysed with RIPA lysis buffer (Meilunbio, China) for protein extraction. The supernatants were collected for protein analysis after the lysates were centrifuged at 14,000 × g for 15 min at 4°C. Protein samples were separated via 10% SDS-PAGE (Meilunbio, China) and transferred to PVDF membranes (0.2 μm, Millipore, Bedford, MA, USA). After probing with rabbit anti-LTF (1:500, BOSTER, China) and anti-β-actin (1:1000, Proteintech, China) overnight at 4°C, the membranes were incubated with goat anti-rabbit IgG (10000, Immunoway, USA) for 1 h. Protein bands were detected using an ECL kit (Meilunbio, China). Densitometry was performed with ImageJ software.

The tissue protein samples were extracted using a RIPA lysis buffer (Applygen, Beijing, China, C1053) containing a protease inhibitor cocktail (Roche, South San Francisco, CA, United States, 11873580001). The proteins were separated based on their molecular weight through electrophoresis and then transferred to polyvinylidene difluoride membranes (Amersham Biosciences). After blocking, the membranes were incubated with anti-COL1A2 (Immunoway, YT6135, 1:1000 dilution) or anti-α-SMA (ABclonal, A7248, 1:1000 dilution). The membranes were then washed 3 times and incubated with goat anti-rabbit IgG secondary antibodies. Blots were then developed with an ECL kit (Meilunbio, Dalian, China, MA0186) and visualized with a chemiluminescence detection system (Syngene, GeneGnome XRQ, Cambridge, UK). Quantitation was performed by scanning and analyzing the intensity of the hybridization bands.

Cells were lysed with radioimmunoprecipitation (RIPA) buffer containing phenylmethylsulfonyl fluoride (PMSF) (Solarbio, Beijing, China) and PhosSTOP™ (Merck, Darmstadt, Germany). Proteins were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. The PVDF membranes were blocked with TBST containing 5% skim milk and incubated overnight at 4 °C with the MMP9 (NO. 13667), HK2 (NO. 2867) and LDHA (NO. 3582) antibodies from Cell Signaling Technology (MA, USA), and N-cadherin (NO. AF4039), vimentin (NO. AF7013) and SCD1(NO. DF13253) antibodies from Affinity Biosciences (OH, USA), and E-cadherin (NO. 20874–1-AP) and ACLY (NO. 15421–2-AP) antibodies from Proteintech (Wuhan, China). Thereafter, the membranes were incubated with the horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature for 1 h, and the signals were visualized with an enhanced chemiluminescence ECL kit (Meilunbio, Dalian, China).

The mouse heart tissues were homogenized in 0.5 ml of RIPA (1:1000 PMSF, Solarbio Science, Beijing) buffer using small tubes and vibrated every 10 min for 5 s at 4°C, and the above process was repeated four times. Solubilized proteins were centrifugated at 13,500 rpm for 25 min, and the supernatant was then collected. The protein concentration of each sample was quantified using the BCA Protein Assay kit (Beyotime, Shanghai). Protein lysates of each group were separated by electrophoresis with SDS-PAGE and electro-transferred onto a PVDF membrane (Millipore). Non-specific proteins on membranes were blocked with 5% non-fat dried milk for 2 h at room temperature, and the membranes were incubated overnight with the following primary antibodies (at a 1:1000 dilution, 4 °C): PRKG1, Ppm1k, Pltp, Hacd2, (ABclonal Technology, Wuhan, China); Pir (Abcam Technology, USA); Thbs1, (Cell Signaling Technology, Danvers, MA); Tmem70, β-tubulin (Santa Cruz Biotechnology). Then, the membranes were incubated with anti-mouse/anti-rabbit IgG antibody (ABclonal Technology, Wuhan, China) at a 1:10000 dilution for 1 h at room temperature. The specific complex was determined by an enhanced chemiluminescent (ECL) kit (Meilun, Dalian, China) and a multiplex fluorescent imaging system (ProteinSimple, CA).

The proteins from Kasumi-1 and HL-60 cells treated with sulfasalazine were lysed in Radio Immunoprecipitation Assay (RIPA) buffer containing phosphatase and protease inhibitor. After centrifugation at 4 °C, the proteins in the supernatant were collected and the protein concentration was determined by bicinchoninic acid (BCA) protein kit (Sigma, #71285-M). Forty micrograms of protein were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. Subsequently, the PVDF membranes were blocked with 5% non-fat milk in TBST (TBS buffer containing 0.1% Tween 20) to prevent nonspecific binding and incubated with primary antibodies GAPDH (ImmunoWay, #YN5585) and SLC7A11 (Abcam, #ab175186) overnight at 4 °C. After washing with TBST, the membranes were incubated with anti-horseradish peroxidase-linked IgG secondary antibody (ImmunoWay, #RS0002). Blots were developed with an ECL kit (Meilunbio, #MA0186).

Proteins were extracted in RIPA lysis buffer supplemented with protease inhibitors. The concentration of lysed proteins was measured using a BCA kit (Thermo Fisher Scientific Inc., MA, USA). Then, proteins were separated via 10% or 12% SDS-PAGE and transferred onto PVDF membranes (Sigma, St. Louis, MO, USA). The membranes were blocked by Quick Blocking Solution (Biyuntian Biotechnology Co., Ltd., Shanghai, China) for about 10–15 min before incubation with primary antibodies and secondary antibodies. An ECL kit (Dalian Meilun Biotechnology, Dalian, China) was used to detect the blots, and the target protein signals were quantified using ImageJ software.

western-blot assays were performed according to the standard procedure to analyze protein expression in cells. Primary antibodies were used for assays: TAZ (Proteintech, 23306-1-AP), USP26 (sigma, SAB1300266), HA (Proteintech, 51064-2-AP), Myc (Proteintech, 60003-2-Ig), Flag (Proteintech, 66008-1-Ig), and GAPDH (Proteintech, 60004-1-Ig). Protein signals were visualized using an enhanced chemiluminescence (ECL) kit (Meilun, China) and detected by ChemiDoc XRS + Imaging System (Bio-Rad).