TMT-based p62 interactome analysis demonstrates binding of p62 to mitochondria in leukemia cells. (A) Experimental scheme. (B) Identification of interactors by quantification of proteins in WT samples compared with a p62^−/− cell line as control. Fold change (FC) [log2] is plotted against corrected P values (Benjamini Hochberg, FDR 0.05). Significant hits (P values <.05 and FC >0.5 log2) are separated by dotted lines. Significantly enriched proteins are considered interactors. (C) Volcano plot as in panel B with all identified mitochondrial proteins is indicated in red. (D) Venn diagram demonstrating the distribution of mitochondrial and nonmitochondrial proteins enriched by p62 immunoprecipitation. (E) Heat map of all identified mitochondrial interactors, with scaled intensities per protein across all replicates and treatments. (F) Heat map of interactors not localizing to mitochondria with scaled intensities for all replicates and treatments. (G) Coimmunoprecipitation validation of interaction between p62 and identified interactors in steady state and after 3 hours of treatment with 100 nM BafA. Lysates were analyzed by western blotting for p62, mitochondrial interactor GBAS, and nonmitochondrial interactor TMEM160. Total cell lysate (TCL) was used as an expression control. (H) Mitochondrial fractions were isolated from WT and p62^−/− MN1-driven ldMBM leukemia cells. Lysates from mitochondria (MT) and TCL were analyzed by western blotting for p62 and mitochondrial marker VDAC1, COXIV. β-Actin served as a loading control. Experiments (G-H) were performed in triplicate. IgG, immunoglobulin G.