SAMM50 is important for basal mitophagy. (A and B) Lysates from WT and SAMM50 KD HeLa cells left untreated or treated with either BafA1 or MG132 for 24 h, respectively, were immunoblotted with indicated antibodies (A) and quantified (B). Values are mean ± SD from three different experiments. **, P < 0.005; *, P < 0.01; †, NS; one-way ANOVA. (C) Lysates from HeLa cells treated with a combination of Pepstatin A and E64d for 24 h to block lysosomal protein degradation were immunoblotted using the indicated antibodies. (D and E) WT, DRP1 KO (D), and MUL1 KO (E) cells were treated with BafA1 for 24 h followed by immunoblotting with indicated antibodies. (F and G) WT and PINK1 KO cells were left untreated or treated with either BafA1 for 24h (F) or a combination of OA for 3h (G). Indicated protein levels were analyzed by immunoblotting. (H) WT, SAMM50 KD, and SAMM50 KD HeLa cells reconstituted with Myc-SAMM50 WT and Myc-SAMM50 Δ1-40 mutant were either untreated or treated with BafA1 for 24 h. Lysates were prepared and used for immunoblotting with the indicated antibodies. (I) Densitometric analysis of relative protein levels for (H) from three independent experiments. Values are mean ± SD. **, P < 0.005; †, NS; one-way ANOVA. (J and K) Mapping of binding site on SAMM50 for MIC19, MTX1, MTX2, and p62. (J) Myc-tagged proteins were in vitro translated in the presence of radioactive methionine and used in GST-pulldown assay with GST-tagged SAMM50 WT and indicated mutants. (K) In vitro translated SAMM50 WT and indicated mutants with GST and GST-tagged MIC19, MTX2, and p62. Bound Myc-tagged proteins were detected by AR while GST proteins were stained with CBB.