0.22 μm polyvinylidene fluoride membrane

A549 and SPC-A1 cells were lysed with RIPA extraction reagent (Beyotime) supplemented with a protease inhibitor cocktail (Roche). Proteins in cell lysates were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to 0.22 μm polyvinylidene fluoride membranes (Millipore). Membranes were probed with specific antibodies using standard methods. Specific protein bands were detected by incubation with ECL chromogenic substrate and quantified by densitometry (Quantity One software; Bio-Rad, Hercules, CA, USA). Antibodies against E-cadherin, N-cadherin, Vimentin, and GAPDH (1:1000) were purchased from Cell Signaling Technology; antibodies against VAV3, EZH2, LSD1, Ago2, and HuR were purchased from Millipore; antibody against BTG2 was purchased from Absin. GAPDH was probed as an internal control. Antibodies are listed in Additional file 1: Table S1.

Protein extraction was performed as follows: after treatment with DCMPI (30 μg/mL, 60 μg/mL) for 24 h, HT-29 cells were cultured, collected, rinsed twice with ice-cold PBS, lysed, incubated in RIPA buffer containing a 1% protease inhibitor cocktail for 30 min on ice, and then centrifuged at 12,000 × g for 15 min. The supernatants were harvested and prepared by mixing with 5× sample buffer for subsequent Western blot analysis. The protein samples were then separated by 10% SDS-PAGE and transferred onto 0.22 μm polyvinylidene fluoride membranes (Millipore, Bedford, MA, United States). The membranes were blocked with 5% non-fat skim milk for 1 h at room temperature and then incubated with the appropriate primary antibodies overnight at 4°C. The membranes were incubated with a horseradish peroxidase-conjugated secondary antibody at room temperature for 2 h, followed by rinsing three times with 1× TBST. The protein bands were visualized utilizing ECL detection reagents (Bio-Rad, United States).

Cell protein lysates were separated by 10% sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, transferred to 0.22 μm polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA), and incubated with specific antibodies. ECL chromogenic substrate was used for quantification by densitometry (Quantity One software; Bio-Rad, Hercules, CA, USA). GAPDH antibody was used as a control. Anti-P21 and anti-EZH2 were purchased from Cell Signaling Technology (Boston, MA, USA).

Cell proteins were extracted with RIPA lysis buffer (Beyotime, Beijing, China). The protein concentrations were determined by bicinchoninic acid (BCA) protein assay kit (Cwbio, Jiangsu, China), and then boiled at 95°C with 6 × loading buffer for 8 min. Samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immediately transferred onto 0.22 μm polyvinylidene fluoride membranes (Millipore, USA), the membranes were blocked with Tris-buffered saline with Tween 20 (TBST) containing 5% nonfat milk at room temperature for 60 min. After washing three times, the membranes were incubated overnight at 4°C with anti-TLR4 (1:1,000 dilution, AF7017, Affinity). Then blots were bound with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:20,000 dilution, Proteintech) for 60 min at 37°C. After washing again with TBST three times, experimental results were obtained by the pierce enhanced chemiluminiscent (ECL) western blotting kit (Thermo Scientific), and imaged by chemiluminescence system (BIO-RAD, USA).

Protein samples were prepared by homogenizing cardiac tissue in radioimmunoprecipitation assay (RIPA) buffer containing proteinase and phosphatase inhibitors (Complete and PhosSTOP; Roche, Basel, Switzerland). Protein extracts from myocardial tissue were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to 0.22 μm polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). The membranes were then blocked in Tris-buffered saline with 0.1% Tween 20 containing 5% nonfat dry milk for 2 h at room temperature. Subsequently, the membranes were incubated overnight at 4 °C with primary antibodies, including Nr4a1 recombinant rabbit monoclonal antibody (Cat#MA5-32647, Thermo Fisher Scientific, 1:1000) and HK2 recombinant rabbit monoclonal antibody (Cat#ab209847, Abcam, 1:1000;). The housekeeping protein GAPDH (Cat#KM9002T, Sungene Biotech, 1:4000) was used as reference protein to normalize the target proteins during western blot analysis. After washing, the membranes were incubated with secondary antibodies antibody IgG (Cat# A0208, Beyotime Biotech, 1:1000) at the appropriate dilutions for 1.5 h at room temperature, and detected using the enhanced chemiluminescence substrate kit. The density of each band was quantified using Quantity One. Three biological repeats were performed for each sample.