Horseradish peroxidase labeled goat anti rabbit igg

After collecting Tregs from each group (n = 15/group), total protein was extracted, and the protein concentration was measured by the bicinchoninic acid method. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed for ~1.5 h. Then, proteins were transferred to a nitrocellulose membrane using a semi-dry method for ~50 min. After the transfer, 5% bovine serum albumin was incubated with the membrane at room temperature on a shaker for 30 min and then incubated overnight with LC3 antibody (Cell Signaling Technology, Danvers, MA, USA) and rabbit polyclonal glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (Hangzhou, China). Tris-buffered saline containing Tween 20 was incubated with horseradish peroxidase-labeled goat anti-rabbit IgG (Cell Signaling Technology) for 1 h at room temperature (20–25°C); electrochemiluminescence detection was performed to evaluate the protein signal by exposure to a gel imager. Protein levels were normalized to GAPDH, and changes were determined such that the LC3-II/GAPDH ratio reflected the degree of autophagic protein expression. LC3-II is more hydrophobic than GADPH, and thus LC3-I appeared as the upper band in the electropherogram, while LC3-II was in the lower band. The ratio of LC3-II/GAPDH was calculated based on GAPDH as the internal reference level of expression for statistical analysis.

The mouse lungs were harvested and homogenized, and then lysed in 500 μl of cold lysis buffer (150 mM NaCl, 50 mM Tris, pH 8.0, 10 mM EDTA, NaF, sodium pyrophosphate, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and 10 μg of leupeptin/ml). Lung homogenates were clarified by centrifugation at 10,000×g, at 4 °C for 10 min, and the clarified lysates were mixed with SDS-PAGE sample buffer (60 mM Tris, pH 6.8, 10% glycerol, and 2.3% SDS) and heated to 95 °C for 5 min. The samples were separated by electrophoresis using a 4–15% gradient gel and transferred to polyvinylidene fluoride (PVDF) membranes. For the detection of proteins, the membranes were immunoblotted with rabbit polyclonal antibody specific for RIG-I (Abcam, Cambridge, MA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; R&D Systems). The membranes were incubated with horseradish peroxidase-labeled goat anti-rabbit IgG (Cell Signaling Technology, Beverly, MA) and chemiluminescent reagents (Pierce Biotechnology, Rockford, IL). Blots were developed using the Syngene G:box Bioimaging System and GeneTools software (Syngene, Frederick, MD) and quantified using ImageQuant software (BD/Molecular Dynamics, Bedford, MA).