Odyssey laser digital imaging system

RAW264.7 cells were washed twice with cold phosphate-buffered saline (PBS, pH = 7.0), and lysed in radioimmunoprecipitation (RIPA) buffer [Cell Signaling Technology (CST), USA] supplemented with protease inhibitors (Roche, USA). The protein concentration of each sample was assayed using the bicinchoninic acid method (BCA kit) (Pierce, Rockford, IL, USA). Equal amounts of protein (60 μg) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 12.5% gel. Then, the protein was blotted onto a NC membrane. After blocking with 5% non-fat milk in 20 mM of tris-buffered saline (TBS) with 0.1% Tween for 1 h at room temperature with shaking, they were incubated with the indicated primary antibodies at 4 °C overnight, followed by the appropriate fluorescent secondary antibodies (1:5000 dilution) for 2 h at room temperature. The immune-reactive proteins were detected using the Odyssey laser digital imaging system (Gene Company). Primary antibodies employed in this study included anti-GAPDH (1:1000, CST, USA), anti-p65 (1:1000, CST, USA), anti-Erk1/2 (1:1000, CST, USA), anti-JNK (1:1000, CST, USA), anti-p38 (1:1000, CST, USA), anti-p-p65 (1:1000, CST, USA), anti-p-Erk1/2 (1:1000, CST, USA), anti-p-JNK (1:1000, CST, USA), anti-p-p38 (1:1000, CST, USA) and anti-CXCR4 (1:1000, Thermo Fisher, USA).

After treatment or stimulation, the lung tissues were lysed by RIPA buffer (Cell Signaling Technology, USA). The concentration of total protein was measured by BCA assay (Pierce) and adjusted to the same concentration with extraction reagent. Samples were heated at 100°C for 5 min and loaded onto 10% SDS-PAGE. After electrophoresis, the gel was transferred onto polyvinylidene fluoride (PVDF) membrane and blocked with 5% BSA. The membrane was incubated with primary Abs and then with the appropriate fluorescent secondary Abs. Subsequently, the immunoreactive proteins were detected using the Odyssey laser digital imaging system (Gene Company, Hongkong, China). The primary antibodies including anti-AKT, anti-phospho-AKT, anti-ERK, anti-phospho-ERK, anti-P38, anti-phospho-P38, anti-P65 and anti-phospho-P65 are all rabbit monoclonal antibodies (Cell Signaling Technology, USA). The P2Y6 antibody is a rabbit polyclonal IgG (Santa Cruz Biotechnology, USA). The secondary Antibody is IRDye^® 680LT Goat anti-Rabbit IgG (LI-COR Bioscience, USA).