Figure 3 from Disruption of KLHL6 Fuels Oncogenic Antigen Receptor Signaling in B-Cell Lymphoma

KLHL6 interacts with BCR and forms homodimers and heterodimers that are differentially impacted by recurrent mutations. A, High-throughput proteomic approach to determine KLHL6 protein interactions and impact of recurrent mutations in DLBCL context. C-terminally tagged KLHL6 constructs encoding WT protein and different mutants or EmGFP (tagged and nontagged) controls were used in 4–8 biological replicate purifications in two batches of mass spectrometry. B, Volcano plot of DESeq2 model between WT KLHL6 and EmGFP pull-downs showing only the KLHL6 interacting proteins (positive fold-change). Labeled gene names of proteins denote high-confidence interactors detected by SAINT analysis. Plots on the right highlight interacting protein components of the Cop9-signalosome, immune proteasome, and BCR signaling. C, Representative IF microscopy images showing colocalization between endogenous KLHL6 and C-terminally V5-tagged KLHL7 and KLHL26 constructs. Scale bar, 10 µm. D and E, Volcano plots of differentially interacting proteins between WT KLHL6 (eight replicates) and (D) KLHL6L65P (four replicates) and (E) KLHL6L90F (eight replicates). Proteins with positive fold-change (log₂) interact more strongly with the WT bait. F, Box plots of spectral counts of peptides from selected prey proteins in the pull-downs according to different baits. Dots represent biological replicates. Mann–Whitney U test for comparisons, P values shown. G and H, Bar and dot plots of spectral counts for specific peptides in BTB mutant KLHL6 pull-downs encoding either mutated or WT amino acids from (G) KLHL6L65P and (H) KLHL6L90F peptides. Dots represent individual replicates, and bars represent their means. All analyzed pull-downs contained mutant (bait) and WT (prey) peptides. I, Western blot analysis of SuDHL4 cells transduced with Strep-tagged WT and mutant KLHL6 constructs or EmGFP control. In the eluate, purified for Strep-tag, one protein (bait) is detected with HA-antibody, whereas two proteins (bait and prey) are detected with KLHL6 antibody confirming homodimerization. J, Balloon plot showing the interactors of WT and mutant KLHL6 baits according to SAINT analysis. K, Balloon plot of WT KLHL6 interactors compared with EmGFP control purifications identified with AP-MS across multiple lymphoma cell lines from different molecular backgrounds. Validated key interactors identified from SuDHL5 are shown.