Figure 4 from Disruption of KLHL6 Fuels Oncogenic Antigen Receptor Signaling in B-Cell Lymphoma

Complementary interactome screening with Kelch-domain coupled proximity-labeling reveals potential substrates of KLHL6 in B-cell context. A, Schematic representation of proximity-labeling (PL) substrate screening with BioID2 approach for C-terminally tagged KLHL6. B, Volcano plot of the DESeq2 model showing the differentially interacting proteins between BioID2-tagged WT KLHL6 and EmGFP constructs showing only the KLHL6-interacting proteins with a positive log₂-transformed fold-change (LFC). Colors as in Fig. 3B. C, Comparison of KLHL6 interactomes obtained with the complementary protein interaction screening methods. Evidence for protein binding by APs (“AP-MS”) and proximity-labeling (“BioID2”) are indicated with LFCs of DESeq2 models with respective EmGFP control pull-downs. Labels indicate proteins with strong evidence by both methods and AP/PL-specific interactors not considered common contaminants in mass spectrometry experiments. D, Summary of identified KLHL6 interactors with the complementary screening methods. E, Western blots showing the impact of KLHL6 expression manipulation at the protein levels of BCR components CD79A, CD79B, and IgH (IgM or IgG) in four different cell lines. Below, bar blots show the quantified protein levels of the BCR components in cells with ectopic KLHL6 overexpression (KLHL6 OE) or KLHL6 silencing (KO, knockout; KD, knockdown) normalized to the total protein levels in relation to their respective controls (EmGFP, sgCTRL, or sh_CTRL). F, Western blots showing the impact of KLHL6 expression manipulation on the protein levels of its proximity interactors BANK1 and LuZP1. Heatmaps show the average quantified protein levels from KLHL6 overexpressing or knockout cells normalized to β-actin in relation to the EmGFP- or sgCTRL-transduced cells.