Gel pro analyzer software version 4

Lysates of the cells were prepared using a radioimmunoprecipitation assay buffer mixed with phenyl methylsulfonyl fluoride (Beyotime Institute of Biotechnology). The lysates were centrifuged for 10 min at 12,000 x g at a temperature of 4˚C. Concentrations of the proteins were determined using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology). The proteins (20 µg samples/lane) were separated by SDS PAGE on 12% gels and subsequently electro-transferred using electro-blotting apparatus (Bio Rad Laboratories, Inc.) onto nitrocellulose membranes. The membranes were blocked with tris-buffered saline with 1/1,000 tween containing 5% non-fat milk for 1 h at room temperature. Subsequently the membranes were incubated overnight with antibodies against RbAp48 (cat. no. ab79416; 1:1,000; Abcam), NF-κB (cat. no. 545380-34-5; 1:1,000; Merck KGaA) and GAPDH (cat. no. D16H11; 1:1,000; Cell Signaling Technology, Inc.) at 4˚C. After washing, membranes were incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibodies (1:2,000; cat. no. 7074; Cell Signaling Technology, Inc.). After incubation, membrane washing was performed with TBS and Tween-20 followed by enhanced chemiluminescence reagent (EMD Millipore) treatment. For analysis, Gel Pro Analyzer software version 4.0 (Media Cybernetics, Inc.) was used.

Radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, China) was adopted to isolate protein from lung tissues. Then, protein fractions were separated on 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by transferring to polyvinylidene difluoride (PVDF) membranes. The membranes were closed with 5% milk and incubated with TLR4 (1: 1000; Cell Signaling Technology, Danvers, MA, USA), MyD88 (1: 1000; Cell Signaling Technology), p-p65 (1: 1000; Cell Signaling Technology), p65 (1: 1000; Cell Signaling Technology), p-p38 (1: 1000; Cell Signaling Technology), p-p38 (1: 1000; Cell Signaling Technology), and GAPDH (1: 1000; Cell Signaling Technology) antibodies overnight at 4°C. Then the membranes were coupled with second antibody (1: 2000; Cell Signaling Technology) for 1 hour at room temperature. At last, the proteins were detected using enhanced chemiluminescence (Beyotime, Shanghai, China) with Canon imaging system and quantified by using Gel-Pro-Analyzer software version 4.0 (Media Cybernetics, Inc., Rockville, MD, USA).

Proteins were extracted using radioimmunoprecipitation assay lysis buffer and protein concentration was measured using a Bicinchoninic Acid Protein Assay kit (both Beyotime Institute of Biotechnology, Shanghai, China). Subsequently, 40 µg protein in each group were separated by 8, 10 or 12% SDS-PAGE and the separated proteins were transferred onto polyvinylidene fluoride (PVDF) membranes (EMD Millipore, Billerica, MA, USA). The PVDF membranes were blocked with 5% skim milk or 1% bovine serum albumin (Biosharp, Hefei, China) at room temperature for 1 h, and then incubated with FOXC1 (1:1,000), N-cadherin (1:1,000), E-cadherin (1:1,000), Vimentin (1:1,000), Snail (1:1,000), Twist (1:1,000), β-catenin (1:1,000), p-β-catenin (1:1,000), c-myc (1:1,000) and GAPDH antibodies (1:1,000) overnight at 4°C. The PVDF membranes were rinsed and then incubated with corresponding horseradish peroxidase-labeled secondary antibodies (cat. no. A0208; 1:5,000; Beyotime Institute of Biotechnology) for 45 min at 37°C. Subsequently, the PVDF membranes were visualized using an enhanced chemiluminescent kit (Beyotime Institute of Biotechnology). The target bands were scanned and analyzed by Gel-Pro-Analyzer software version 4.0 (Media Cybernetics, Inc., Rockville, MD, USA).