Quantitative affinity proteomics identifies SQSTM1/p62 as an interaction partner of the CUL3 adaptor SHKBP1. (A) Workflow of the first set of proteomics experiments using the neddylation inhibitor MLN4924 to enrich unstable CRL targets (left) and cartoon representation of drug treatment mechanism (right). Cells were treated with MLN4924 (10 μM) or vehicle for 16 h. (B) Workflow of the second set of proteomics experiments using tandem UBA domain fusions to SHKBP1 to enrich ubiquitinated substrates (left) and model of ligase trap (right). Cells stably expressing the corresponding construct were treated with MG-132 (20 μM) for 2 h. (C) Workflow of the third set of proteomics experiments using the SHKBP1^F44A CUL3 binding–deficient mutant to reduce CRL complex components in IP compared with SHKBP1^WT (left) and corresponding model (right). (D–F) Volcano plots from the three SILAC MS proteomics experiments, showing log₂ (fold changes of protein abundance in heavy/light samples) vs. statistical significance (–log₁₀ [P value]). Proteins whose change was below the cutoff (fold change < 1.2) are indicated in gray. Those above the cutoff with P values above 0.05 are shown in green, and those with P values below 0.05 are shown in purple.