Expression of Autophagy-Related Protein LC3-II Detected by Western Blotting

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.