IP-MS and TurboID-MS confirm the interaction between FCRL3 and UBASH3A. (A) Schematic representation of FCRL3 and the C-terminally tagged FCRL3-3xFLAG protein. The 3xFLAG tag is shown as green squares. (B) Surface (left) FCRL3 staining and intracellular (right) anti-FLAG staining showing surface expression of the FCRL3-3xFLAG construct. (C) Western blot showing efficient immunoprecipitation of FCRL3-3xFLAG with anti-FLAG agarose beads in Jurkat cells expressing LV-FCRL3 and LV-FCRL3-3xFLAG. Immunoblot was performed using an anti-FCRL3 antibody. (D) Scatter plot showing the enriched proteins in the IP-MS of the FCRL3-3xFLAG compared with untagged FCRL3. In this experiment, to enhance the separation of true interactors from experimental noise, an additional filter was applied by selecting proteins present in at least 3 out of 4 replicates of the FCRL3-3xFLAG condition and in <2 replicates of the untagged control (FCRL3), followed by imputation of the missing values. A two-sided two-samples t test (0.01 permutation-based FDR cut-off, 250 randomizations) was employed to identify significant changes, and the results were visualized with a volcano plot generated with R, version 4.4.2. (E) Schematic representation of the Protein A-TurboID experimental workflow for UBASH3A. The anti-UBASH3A antibody was added to Jurkat cells ectopically expressing FCRL3 or the EGFR-FCRL3 140 aa chimera (or control cells), followed by the addition of Protein A-TurboID fusion protein and biotin. After streptavidin pull-down, biotinylated proteins were analyzed by mass spectrometry. (F) Differentially enriched proteins in the FCRL3-transduced cells versus control (left) and chimera-transduced cells versus control (right). N = 4 independent samples. Source data are available for this figure: SourceData FS5.