E cadherin antibody

All the chemical reagents were purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China) and used without further purification or modification unless otherwise specified. Fetal bovine serum (FBS), RPMI 1640 medium, DMEM medium, and trypsin–EDTA were purchased from Gibco-Brl (Grand Island, NY, USA). Rabbit anti-mouse or anti-human PD-L1 antibody, E-cadherin antibody, Vimentin antibody, goat anti-rabbit IgG HRP and β-actin antibody were purchased from Affinity Biosciences Inc (USA), AMPK antibody, pAMPK antibody, and TGF-β antibody were purchased from Cell Signaling Technology (USA). All biochemical reagents were used without further purification.

The lung tissue samples of mice in each group were crushed, lysed, and centrifuged. The total protein concentration was measured with a BCA kit, followed by SDS-PAGE electrophoresis, and then transferred to a PVDF membrane. At the end of the transfer membrane, blocking was performed at room temperature for 1 h with 5% nonfat dry milk, and then, the membrane was incubated at 4°C overnight with corresponding primary antibodies (E-cadherin antibody (Affinity, AF0131), collagen I antibody (Affinity, AF7001), vimentin antibody (Affinity, AF7013), N-cadherin antibody (Affinity, AF4039), a-SMA antibody (Affinity, BF9212), TGF-β1 antibody (Affinity, AF1027), TβRI antibody (Affinity, DF7309), TβRII antibody (Affinity, DF13307), phospho-Smad2 (Ser250) antibody (Affinity, AF3450), Smad2 antibody (Affinity, AF6449), phospho-Smad3 (Ser423 + Ser425) antibody (Affinity, AF8315), Smad3 antibody (Affinity, AF6362), and Smad7 antibody (Affinity, AF5147)). After incubation with an HRP-labeled secondary antibody at room temperature for 1 h, the immunoreactive bands were visualized by developing solution. After scanning, the gray value of each band was determined by image analysis software, and the ratio with the internal reference β-actin was used as the basis for semiquantitative analysis.

Western blot analysis detected the protein expression of ZO-1 and E-cadherin. The cells stimulated by antimicrobial peptides were treated by 0.25 ml lysis buffer (Solarbio, Peking, China) with 1 mM phenylmethanesulfonyl fluoride (PMSF). The supernatants were collected by centrifugation at 10000 × g for 5 min at 4°C. The protein samples (20 μg) were electrophoretically separated in 10% SDS-PAGE gels. Then, the protein samples were transferred to Nitrocellulose (NC) membranes. After blocking for 1 h at room temperature, the membranes were incubated overnight at 4°C with the following primary antibodies: anti-ZO 1 antibody (Affinity, Cincinnati, OH, United States), E-cadherin antibody (Affinity, Cincinnati, OH, United States), and anti-beta Actin antibody. The NC membranes were washed and incubated with a secondary antibody (Abcam, Cambridge, England) for 1 h at room temperature. Finally, chemiluminescence detection was performed with Immobilon ECL Ultra Western HRP Substrate (Millipore, Massachusetts, United States) on a MicroChemi imaging system (DNR, Israel). The intensity of each band was quantified in ImageJ software. Each band was standardized by internal reference gene (Beta Actin), and then compared with the control group.