Protein extraction and immunoblotting were performed as described previously [20 (link)]. Briefly, proteins were extracted from the tissue using a standard extraction reagent supplemented with a protease inhibitor (KANGCHEN; Shanghai, China). Protein concentrations were determined using a BCA protein assay kit (Beyotime Institute of Biotechnology; Haimen, China). Proteins were separated using SDS-PAGE, electrotransferred to nitrocellulose membranes and incubated with a primary antibody for 8–12 h at 4 °C (Table 3 ). The samples were then incubated with an IRDye800CW-conjugated secondary antibody (Rockland; Gilbertsville, PA, USA) for 1 h at 25 °C. Images were acquired with the Odyssey infrared imaging system (Li-Cor Bioscience; Lincoln, NE, USA). All immunoblotting experiments were repeated at least three times.
Primary antibodies used in Western blots.
| Antibody | Molecular weight (kDa) | Dilution |
|---|---|---|
| Beclin-1 (Cell Signaling Technology) | 60 | 1:500 |
| LC3 (Novus Biologicals) | 14/16 | 1:500 |
| p62 (Cell Signaling Technology) | 62 | 1:500 |
| p-AMPK (Thr172) (Cell Signaling Technology) | 62 | 1:500 |
| AMPK (Cell Signaling Technology) | 62 | 1:500 |
| p-mTOR (Ser 2448) (Cell Signaling Technology) | 289 | 1:500 |
| mTOR (Cell Signaling Technology) | 289 | 1:500 |
| p-p70S6K (Ser371) (Cell Signaling Technology) | 70 | 1:500 |
| p70S6K (Cell Signaling Technology) | 70 | 1:500 |
| GAPDH (Beyotime Biotechnology) | 36 | 1:1000 |