Fig. 2

ID4 binds to MDC1 and interacts with the BRCA1 network. a Schematic of ID4 and IgG RIME data analysis; ID4 and IgG immunoprecipitations were conducted in technical duplicate or triplicate, and in biological triplicate. All proteins identified in the IgG controls (for each biological replicate) were removed from each of the ID4 IPs as non-specific, background proteins. The remaining proteins were compared across technical replicates to generate a list of medium-confidence proteins that were robustly identified in > 1 ID4 RIME technical replicate. The biological replicates were then compared to generate a list of targets present in > 1 biological replicate in an individual cell line (i.e., > 6 technical replicates conducted over three biological replicates). This resulted in the identification of 22 (HCC70), 21 (HCC1954), 21 (MDA-MB-468) and 30 (OVKATE) proteins. Targets from the different cell lines were compared, identifying a list of ID4 interactors present across multiple cell lines. b Venn diagram showing comparison of the high-confidence targets identified in all four cell lines: HCC70, HCC1954, MDA-MB-468 and OVKATE. Common targets across the four cell lines are indicated. Overlap generated using Venny [58]. c Top six highest enriched gene sets identified through Gene Set Enrichment Analysis (GSEA) of the ID4 proteome (1106 proteins) identified in (b) (all proteins identified in ID4 RIME and not in IgG RIME), compared to C2 (curated gene sets), C4 (computational gene sets), C6 (oncogenic signatures), C7 (immunologic signatures) and H (hallmark gene sets) gene sets. Proteins identified in > 1 technical replicate of ID4 RIME (and not in IgG RIME) were considered in GSEA analysis. d, top: Immunohistochemistry analysis of ID4 protein expression across HCI001, HCI002 and HCI009 triple-negative PDX models, 100 μm scale, bottom; SWATH proteomic analysis of ID4 and IgG RIME conducted on HCI001, HCI002, HCI009 PDX models. Heatmap showing quantification of ID4 and MDC1 abundance, both significantly differentially expressed proteins in ID4 RIME compared with control IgG RIME (p value < 0.05 and fold change > 2). RIME and SWATH were conducted on the same sample, one biological replicate per tumour. 100 μm scale. e Immunoprecipitation was conducted on MDA-MB-468 cells prepared using the RIME protocol. Top: Input control and IgG (mouse and rabbit) compared to IP with anti-ID4 antibodies and MDC1. Western blotting is shown using an independent monoclonal MDC1 antibody. Bottom: Input control and IgG rabbit compared to IP with anti-ID4 antibodies and MDC1. Western blotting is shown using an independent monoclonal ID4 antibody