Pab36264

Proteins were extracted from cells, and their concentration was measured using a bicinchoninic acid protein assay kit (Beyotime, China). Total protein was separated by 8% or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. The following primary antibodies were used: osteopontin (OPN, 1 : 1000, PB0589, Boster), focal adhesion kinase (FAK, 1 : 500, BM4303, Boster), p-FAK (1 : 500, BM4426, Boster), Ras (1 : 400, BM4281, Boster), mitogen-activated protein kinase (MAPK, 1 : 1000, BM4439, Boster), p-MAPK (1 : 2000, P00176, Boster), phosphatidylinositol 3-kinase (PI3K, 1 : 1000, BM5187, Boster), p-PI3K (1 : 1000, ab182651, Abcam), and GAPDH (1 : 1000, PAB36264, Bioswamp). After three washes with phosphate-buffered saline/Tween 20, the membranes were incubated with horseradish peroxidase-conjugated secondary goat anti-rabbit IgG (1 : 20000, PAB160011, Bioswamp). Protein bands were visualized by enhanced chemiluminescence color detection (Tanon-5200, TANON) and analyzed using AlphaEase FC gel image analysis software.

BEAS-2B cells or human lung tissue samples were lysed using the protein isolation kit (#KGP250, KeyGEN BioTECH, Nanjing, China) or homogenized with lysis buffer containing phenylmethylsulphonyl fluoride on ice, followed by centrifugation at 12,000 rpm for 5 min. Protein concentrations were measured using a BCA quantification kit (Keygentec, Nanjing, Jiangsu, China). Proteins were separated on 10% SDS gel and transferred to a polyvinylidene fluoride membrane, followed by 1 h of blocking with 5% skim milk. The membrane was then incubated with anti-N-cadherin (1:100; ab18203; Abcam), anti-vimentin (1:5000; ab92547; Abcam), anti-α-SMA (1:500; ab7817; Abcam), anti-E-cadherin (1:1000; ab76055; Abcam), or anti-GAPDH (1:10,000; PAB36264; Bio swamp, Wuhan, Hubei, China) antibody overnight at 4 °C, followed by 3 washes with Tris-buffered saline containing 0.1% Tween 20 (TBST). The membrane was then incubated with horseradish peroxidase-conjugated secondary antibody (1:10,000; SAB43710 or SAB43711; Bio swamp) for 2 h at room temperature. After an additional three washes with TBST and the addition of exposure solution (Millipore, #WBKLS0500, USA), the protein bands were visualized using an enhanced chemiluminescence reagent (Life iLab, Shanghai, China) and analyzed using the Image J software (NIH, Bethesda, MD, USA).