NOTCH2 is a specific interactor of KLHL6. (A, left) HLY1-TurboID-KLHL6 was treated with DMSO or 5 μM MLN4924 for 6 hours; biotin was added at 5 μM for 6 hours. The graph is a scatter plot showing the log₂ ratio between the unique peptides identified via mass spectrometry upon biotin AP in the MLN4924 and DMSO condition. The blue circles represent the KLHL6 interactors enriched by at least threefold. (A, right) Scatter plot showing the log₂ ratio between the unique peptides identified via mass spectrometry in the FLAG immunoprecipitation (FLAG-IP) of KLHL6 and the empty vector (EV).²⁵ The red circles represent the KLHL6 interactors enriched by at least fourfold. (B) Venn diagram showing the overlap of the KLHL6 interactors that were identified in panel A. (C) Immunoblot analysis for the indicated proteins from the whole cell lysate (WCL) of U2932, OCI-LY10, SUDHL6, and SUDHL10 cells stably expressing Cas9 and 2 sgRNAs against KLHL6 and control sgRNA, (D) Immunoblot analysis for the indicated proteins from the WCL and FLAG-immunoprecipitated (FLAG-IP) samples of HEK293T cells overexpressing the indicated FLAG-tagged BTB proteins and F-box protein. The cells were treated with MLN4924 for 6 hours before harvesting. (E) Immunoblot analysis for the indicated proteins from WCL and FLAG- IP samples of U2932, OCI-LY10, SUDHL6, and SUDHL10 cells stably expressing EV, FLAG-KLHL6 (WT) or FLAG-KLHL6 (F97L). (F) Immunoblot analysis for the indicated proteins from WCL and FLAG- IP samples of FLAG-taggedWT KLHL6 and KLHL6 mutant lacking the Kelch domain (ΔKelch). The cells were treated with MLN4924 for 6 hours before harvesting. Unless otherwise noted, immunoblots are representative of 3 independent experiments. gCTRL, control sgRNA.