Fig. 4: Co-immunoprecipitation followed by label-free proteomics confirms that mitochondrial proteins are predominant interactors of p62 in polysomic cells.

a Schematic depiction of experimental procedure for p62 pull-down followed by mass spectrometry. Image partly created in BioRender. b Volcano plots showing log₂-transformed fold change of enriched p62 interactors in WT, 13/3 and 5/4 cells treated with BafA1, highlighting mitochondrial (magenta), karyotype-independent (cyan) and polysomic-specific (orange) interactors. Data is generated from mass spectrometry analysis of n = 4 biological replicates (see Supplementary Fig. 1g, h, and Supplementary Data 3). c Representative immunoblot and d Quantification of p62 expression levels in the diploid parental HCT116 cells (WT-Ctrl.), parental cells transiently overexpressing EGFP (WT-EGFP OE) or p62-EGFP (WT-p62-EGFP OE), and the polysomic 5/4 cells. Data is shown as mean ± s.d. fold change to WT-Ctrl from n = 4 independent experiments, and individual replicates are plotted as dots. P values represent two-tailed unpaired Student’s t-test. e Volcano plots showing log₂-transformed fold change of enriched p62 interactors in WT-EGFP OE and WT-p62-EGFP OE cells, treated with DMSO or BafA1, highlighting mitochondrial (magenta), karyotype-independent (cyan) and polysomic-specific (orange) interactors. Data is generated from mass spectrometry analysis of n = 4 biological replicates (see Supplementary Fig. 1i, j, and Supplementary Data 3). f Percentage of mitochondrial proteins in the measured proteome (grey) and the corresponding percentage within the p62 interactome (magenta) of WT, WT-p62-EGFP OE, 13/3 and 5/4 cells. P values represent results of one-sided hypergeometric test, evaluating the differences in representation of mitochondrial proteins relative to the measured proteome.