Proximity Ligation In situ Assay is a Powerful Tool to Monitor Specific ATG Protein Interactions following Autophagy Induction
Fig 5
Effects of Rapamycin, Rotenone and CCCP on ATG8/cargo adapter interactions.
MDA-MB436 cells were cultured for 24 h at 37°C and 5% CO2 then with Rapa (10 μM for 5h), Rot (50 μM for 24h) or CCCP (100 μM for 24h). (A) SQSTM1/LC3B, (B) SQSTM1/GL1, (C) NIX/LC3B and (D) NIX/GL1 P-LISA were performed according to the manufacturer’s recommendations using mouse anti-NIX or anti-SQSTM1 and rabbit anti-GL1 or anti-LC3B antibodies. Nuclei were stained with DAPI. Each picture is representative of a typical cell staining observed in 10 fields chosen at random. (E) Quantification of P-LISA signals (dots/cell and intensity per dot) was performed using the Blobfinder software. Each bar (Mean ± SEM) represents the mean obtained from the quantification of signals observed in about 200 cells chosen randomly in 5 different fields from 3 independent experiments. ****: p≤0.0001, ***: p≤0.0001, and **: p≤0.001, vs control. Scale bar: 20μm.