Finding the LC3-interactors in senescent human A549 cells during influenza A virus infection

To screen age-related regulators of mitophagy, we examined LC3-interacting proteins by proximity-dependent biotin identification. LC3B was cloned into a mammalian expression vector tagged with a mitochondrial OMM-targeted APEX2 (mito-APEX) label for proximity-based identification of LC3-interacting proteins localized or trafficked to mitochondria during influenza virus infection. Senescent A549 cells, transfected with the mito-APEX-tagged LC3 plasmid for 18 hours, revealed mitochondrial localization of LC3. Following 18 hpi and biotin affinity capture, biotinylated proteins were purified and analyzed by mass spectrometry to identify LC3-interacting proteins.