Enhanced electro chemiluminescence reagents

Lung tissues were lysed and homogenized in radioimmunoprecipitation assay buffer with phosphatase inhibitors and protease inhibitors (Beyotime Biotechnology). Each homogenate was centrifuged at 12,000 rpm for 15 min at 4 °C to extract proteins, and the concentrations were determined by BCA protein assays (7780, CST). Equal amounts of protein from each sample were resolved by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membranes. After blocking at room temperature for 1 h, the membranes were incubated overnight at 4 °C with antibodies against p63 (ab53039, Abcam), phosphorylated signal transducer and activator of transcription (p-STAT3; 9145, CST), STAT3 (9139, CST), jagged 2 (JAG2; NBP1-58284, Novus Biologicals, Centennial, CO, USA), and β-actin (4970, CST). After washed three times with Tris-buffered saline with Tween-20, the membranes were incubated with an appropriate HRP-conjugated secondary antibody at room temperature for 1 h. Protein bands were exposed with enhanced electro-chemiluminescence reagents (Beyotime Biotechnology), and the bands were analyzed with an Imaging System (Bio-Rad, CA, USA).