Figure 2.

Possible interactions between SNAI1 and LC3 or SQSTM1/p62. (A,B) Coimmunoprecipitation. Control or starved HeLa cell lysates were immunoprecipitated by anti-LC3 (A) or anti-SQSTM1 (B) antibodies in 30 µL protein G agarose beads. After washing, the bound proteins were analyzed by western blotting using the indicated antibodies. Whole-cell extracts (5% input) were also assessed by western blotting. IgG indicates nonspecific mouse antibody as a negative control. (C,E) Immunocytochemistry. HeLa cells were cultured on coverslips for 24 h and then starved with HBSS for 4 h. After fixing cells with paraformaldehyde, cells were incubated for 24 h at 4 °C with rabbit polyclonal anti-SNAI1 plus mouse monoclonal anti-LC3 (C) or mouse monoclonal anti-SQSTM1 (E) antibodies. After washing, secondary antibodies (FITC-conjugated anti-mouse antibody or TRITC-conjugated anti-rabbit antibody) were applied to cells. The glass slides were mounted using a mounting medium containing DAPI (to stain nuclei), and all images were captured by confocal microscopy (Olympus FV-1000). (D,F) Colocalization of SNAI1 with LC3 (D) and SQSTM1 (F) was quantified using NIH ImageJ software. Data represent the mean (±S.D.) of three independent experiments (** p < 0.01).