Antibody against lc3

For immunohistochemistry, the slides were incubated with antibody against LC3 (1:100, Cell Signaling Technology) at 4°C overnight and stained with diaminobenzidine (DAB) and counterstained with hematoxylin. The slides were then subjected to gradient ethanol dehydration and dimethyl benzene transparent and mounted with neutral resin cover slides. Images were captured using a Nikon (Tokyo, Japan) ECLIPSE 80i.
For immunofluorescence staining, cells cultured on poly-l-lysine-coated cover glasses were washed by PBS once and then fixed with 4% paraformaldehyde for 15 min at room temperature. After washing, the cells were permeabilized by 0.1% Triton X-100 for 15 min at room temperature. Next, the cells were blocked with 2% BSA for 30 min and then stained with rabbit anti-human LC3 antibody (1:200, Cell Signaling Technology) at 4°C for 6 h. After washing by PBS, the cells were probed with Alexa Fluor 594-conjugated goat anti-rabbit IgG (1:200, Thermo Fisher Scientific) at 4°C for 1 h. To display the cell nuclei, 100 μg/mL DAPI was applied to incubate with cells for 15 min at room temperature. After washing, the cells were subjected to an inverted fluorescence microscope (Olympus IX73, Olympus, Tokyo, Japan) for imaging. The LC3 puncta were measured by ImageJ software (NIH). Five individual pictures were analyzed for each kind of cell.

The antibodies against IR, IRS‐1, Akt, β‐actin and Akt (phosphorylated on serine 473) were purchased from Cell Signaling Technology (Beverly, MA, USA). The antibody against LC3 was purchased from Cell Signaling Technology and Thermo Fisher Scientific Inc. (Waltham, MA, USA). The antibody against p62/SQSTM1 (C terminus) was purchased from Progen (Heidelberg, Germany). The antibodies against Atg7 were purchased from Wako (Osaka, Japan). Chloroquine and digitonin were purchased from Sigma‐Aldrich (St. Louis, MO, USA).