Proximity Ligation In situ Assay is a Powerful Tool to Monitor Specific ATG Protein Interactions following Autophagy Induction
Fig 3
Increase of SQSTM1/GL1 and NIX/GL1 interactions quantified by P-LISA in MCF-7 overexpressing FLAG-GABARAPL1-6His and GFP-NIX.
(A) Expression of GL1 protein was analyzed using western blotting in MCF-7 expressing or not FLAG-GL1-6His. (B) MCF-7 Control or MCF-7 FLAG-GL1-6His cells were cultured for 24 h at 37°C and 5% CO2, fixed, permeabilized, blocked with 5% BSA, incubated with mouse anti-SQSTM1 and rabbit anti-GL1 antibodies antibodies overnight at 4°C and then with an Alexa Fluor 555 goat anti-mouse and an Alexa Fluor 488 goat anti-rabbit, respectively, for 1 h. The cells were then analyzed using a confocal microscope (top panel). For P-LISA, the protocol was performed according to the manufacturer’s recommendations using mouse anti-SQSTM1 and rabbit anti-GL1 antibodies (bottom panel). (C) MCF-7 FLAG-GL1-6His cells were cultured for 24 h at 37°C and 5% CO2 and transfected with pEGFP-NIX plasmid and then fixed and permeabilized. P-LISA was performed as precognized by the manufacturer using rabbit anti-GL1 and mouse anti-NIX antibodies. Nuclei were stained with DAPI. Each picture is representative of a typical cell staining observed in 10 fields chosen at random. Scale bar: 20μm.