Mab2605

MDA-MB-231 and A549 cells were seeded on glass coverslips for 48 h. Cells were then fixed with 4% paraformaldehyde at room temperature for 15 min, washed three times with ice cold phosphate-buffered saline (PBS; 8 g/L NaCl [Calbiochem, 567441], 0.2 g/L KCl [Calbiochem, 529551], 1.44 g/L Na₂HPO₄ [Calbiochem, 56547], 0.24 g/L KH₂PO₄ [Calbiochem, 529568], pH 7.4), permeabilized with PBST (PBS containing 1% Triton X-100 [Calbiochem, 9410]) for 30 min, and blocked in solution containing 5% BSA (Sigma-Aldrich, A2153) for 1 h at room temperature. The cells were incubated with primary antibody (anti-ATG5 [Millipore, MAB2605]; anti-ATG12 [Gene Tex, GTX629815]) at 4°C overnight and washed 3 times with TBST, followed by incubation with secondary antibody for 1 h at room temperature. Cells were washed three times with TBST and the slides were mounted with glycerol-gelatin. Nuclei were counterstained blue with DAPI (Invitrogen, P36935). The images were taken by scanning confocal microscope (MPE, Olympus).

In situ PLA was performed to visualize protein-protein interactions in MDA-MB-231 and A549 cells. Briefly, cells were seeded onto 3-cm dishes overnight for adherence with ~80% confluence. Cells were then washed with PBS twice, and fixed with 4% paraformaldehyde for 15 min at room temperature. The fixed cells were permeabilized with PBS containing 1% Triton X-100 (Calbiochem, 9410-OP) for 30 min, subsequently blocked in Blocking Solution (Sigma-Aldrich, DUO82007) at 37°C for 1 h and incubated with primary antibodies (anti-BIRC5 [Cell Signaling Technology, 2808]; anti-ATG5 [Millipore, MAB2605; Gene Tex, GTX113309]; anti-ATG12 [Gene Tex, GTX629815], and anti-ATG16L1 [Millipore, ABC25]) overnight at 4°C. On the following day, cells were washed twice with washing buffer (Sigma-Aldrich, DUO82049), and incubated with PLA probes in a ratio of 1:5 in antibody diluent for 1 h at 37°C. The cells were then incubated with ligation solution at 37°C for 30 min and subsequently with amplification solution at 37°C for 100 min. Duolink in situ mounting medium (Sigma-Aldrich, DUO82040) together with DAPI were added to the cells at room temperature for 10 min. Cell images were captured with a confocal microscope (FV1000, Olympus).