p62 associates with several mitochondrial proteins. (A) Endogenous p62 was immunoprecipitated from HeLa cells followed by mass spectrometry analysis of associated proteins. Only some mitochondrial related proteins and proteins showing specific interaction with p62 are presented here. (B) HEK293 cells were subjected to subcellular fractionation and immunoblotted with indicated antibodies. (C) Diffraction-limited DV microscopy images of HeLa cells co-stained with antibodies to endogenous p62 and SAMM50. Nuclear DNA was stained with DAPI. Boxes indicate enlarged images shown below. White arrows indicate colocalization events. Scale bars, 10 µm. (D) GST pulldown using in vitro translated Myc-tagged p62 in the presence of radioactive methionine with GST-tagged mitochondrial proteins. Myc-tagged protein binding was measured by AR while GST-tagged proteins were stained with CBB. (E) Endogenous SAMM50 was immunoprecipitated from HeLa cells followed by mass spectrometry analysis of associated proteins. (F) Myc-tagged SAMM50 was in vitro-translated and its interaction with GST-tagged mitochondrial proteins was tested in GST pulldown assays and analyzed by AR.