Enhanced chemiluminescence detection kit

Protein was isolated from THCA cells using RIPA buffer (Beyotime, Nanjing, China). Proteins were separated using 10% SDS-PAGE and later transferred onto PVDF membrane (Millipore, Boston, USA). Membranes were blocked with 5% skim milk, then incubated with the primary antibodies anti-OCT4 (Cell Signaling Technology, Danvers, MA, USA), anti-NANOG (Cell Signaling Technology), anti-GLI2 (Abcam, Cambridge, UK), and anti-β-Actin (Proteintech, Wuhan, China) for 8 hours at 4 °C. Horseradish peroxidase-conjugated anti-rabbit antibody (Cell Signaling Technology) was used as the secondary antibody. An enhanced chemiluminescence detection kit (Invitrogen) was used for blot detection.

Cultured RLE-6TN cells were washed and then pelleted in PBS at 670 g for 5 min. Each cell pellet contained 2 × 10⁵ cells. The cell pellet was subjected to protein extraction by using a cell lysis buffer. The lung tissues were homogenized, incubated in the RIPA lysis buffer (Elabscience, China), added to a protease inhibitor cocktail, and then centrifuged to obtain extracts of lung proteins. A BCA protein assay kit (ThermoFisher Scientific, USA) was used to measure protein concentration. The samples were loaded with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride membrane (Millipore Company, USA). The membranes were blocked with 5% skimmed milk in PBS at 37°C for 2 h, and then incubated with the appropriate primary antibodies overnight at 4°C to assess the protein levels of E-cadherin, Fn, and α-SMA (1:1500; SantaCruz Biotechnologies, USA). The membranes were washed and exposed to their respective horseradish peroxidase conjugated goat anti-mouse IgG (1:1000; sc2031; SantaCruz Company, USA) for 2.5 h at room temperature. The labeled band was detected using an enhanced chemiluminescence detection kit (Invitrogen, USA). The loading control was β-actin. The experiment was repeated three times.

Proteins (60 μg) were loaded and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto a polyvinylidene difluoride membrane. The membrane was blocked and then incubated with primary mouse antibody to neuroligin1 (1:1000, SYSY, Göttingen, Germany) at room temperature for 2 h. Then, the membrane was incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (1:4000, ZSGB-BIO, China) for 1 h. The membrane was also blocked and incubated overnight with primary rabbit antibodies to GluA1 (1:1000, Millipore, Billerica, MA), GluA2 (1:1000, Millipore, Billerica, MA), or PSD-95 (1:500, Abcam, Cambridge, UK). Then, the membrane was incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:1000, KPL, Boston, MA). Finally, the membranes were exposed to reagents from the Enhanced Chemiluminescence Detection Kit (Thermo, Waltham, MA) and x-ray film for visualization of protein bands. The intensity of the protein bands was quantified with densitometry. The β-tubulin was used as the loading control.