Fig. 4

Ultrastructure of the autophagosome and recruited proteins. a After density gradient centrifugation, purified autophagosomes from the CL1-5-Q89L cells were collected. The LC3 protein in purified autophagosomes was labeled by anti-LC3 antibody conjugated with immunogold (12 nm) followed by negative staining and investigation under TEM. The circle and the white arrow indicate the location of the immunogold-labeled LC3 protein on the outside of the autophagosome membrane. b After centrifugation, the whole cell lysate of CL1-5-Q89L and -Vector cells were divided into: (1) the fraction of the post-nuclear supernatant (PNS); (2) the fraction of purified autophagosomes (AP). The levels of TIMP1, Rab37, LC3, EEA1 (endosomal marker), and calreticulin (endoplasmic reticulum marker) in PNS and AP fractions were evaluated using specific antibodies by immunoblotting. c Proteins in the purified autophagosome of CL1-5-Q89L cells were determined by LC–MS/MS, followed by KEGG database analysis to identify possible diseases. d Lung cancer CL1-5 cells were treated with serum-free medium for 24 h, and total protein extraction was immunoprecipitated by anti-LC3 antibody. The expression levels of proteins in the precipitates and whole cell lysates were evaluated by immunoblotting using specific antibodies. WCL whole cell lysate