Journal: The Journal of Experimental Medicine

Article Title: FCRL3 is an immunoregulatory receptor that restrains the activation of human memory T lymphocytes

doi: 10.1084/jem.20242474

Figure Lengend Snippet: Intracellular portion of FCRL3 interacts with UBASH3A. (A) Schematic representation of the Protein A-TurboID experimental workflow. An anti-FCRL3 antibody was added to FCRL3-expressing or nonexpressing Jurkat cells followed by the addition of Protein A-TurboID fusion protein and biotin. After biotinylation and extensive washing, biotinylated proteins were recovered using streptavidin-conjugated beads and subjected to mass spectrometry analysis. (B) Example of Protein A-TurboID experiment. After incubation of FCRL3-expressing Jurkat cells (or empty vector control cells) with a mouse monoclonal anti-FCRL3 antibody together with recombinant Protein A-TurboID, biotinylated proteins were recovered by streptavidin pull-down, followed by western blot to confirm enrichment of FCRL3. The antibody used for immunoblot was a rabbit polyclonal anti-FCRL3. (C) Differentially retrieved proteins following FCRL3 Protein A-TurboID and mass spectrometry of FCRL3-expressing versus nonexpressing cells (log 2 FC ≥ |2|; P ≤0.01; N = 5 replicates). (D) Example of FCRL3 IP. Jurkat cells expressing FCRL3 (or empty vector control) were lysed, and 1.5 mg of protein extract was used for IP with a rabbit polyclonal anti-FCRL3 antibody, followed by western blot with the same antibody. Two independent representative FCRL3 IPs are shown. (E) Differentially retrieved proteins following FCRL3 IP-MS of FCRL3-expressing versus nonexpressing cells (log 2 FC ≥ |2|; P ≤0.01; N = 4 replicates). (F) Co-IP of FCRL3 with UBASH3A. HEK cells were transfected with the indicated plasmids, followed by IP of FCRL3 using a rabbit polyclonal anti-FCRL3 antibody and immunoblot for either UBASH3A (top) or FCRL3 itself (bottom). Data are representative of N = 2 independent experiments. (G) Schematic representation of the FCRL3 protein and C-terminal truncations (top). All proteins were efficiently expressed, as shown by western blot (bottom). (H) Heatmap showing the differentially enriched proteins by IP-MS of Jurkat cells transduced with full-length FCRL3 and its truncations. N = 3–4 independent samples. Significant differences between multiple experimental conditions were assessed with an ANOVA multiple-sample test (0.01 permutation-based FDR cut-off, 250 randomizations). Significant proteins were filtered, and Z-score normalization was applied to each protein across all samples. Unsupervised hierarchical clustering was employed to visualize on a heatmap proteins with a positive Z-score value for all replicates of the conditions LV-FCRL3 and LV-FCRL3-93 aa. IP, immunoprecipitation. Data underlying this figure can be found in . Source data are available for this figure: .

Article Snippet: The UBASH3A protein was targeted by incubating the cells with 4 μg of UBASH3A antibody (Proteintech) for 35 min in digitonin buffer.

Techniques: Expressing, Mass Spectrometry, Incubation, Plasmid Preparation, Control, Recombinant, Western Blot, Protein-Protein interactions, Co-Immunoprecipitation Assay, Transfection, Transduction, Immunoprecipitation

Journal: The Journal of Experimental Medicine

Article Title: FCRL3 is an immunoregulatory receptor that restrains the activation of human memory T lymphocytes

doi: 10.1084/jem.20242474

Figure Lengend Snippet: 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: .

Article Snippet: The UBASH3A protein was targeted by incubating the cells with 4 μg of UBASH3A antibody (Proteintech) for 35 min in digitonin buffer.

Techniques: Protein-Protein interactions, Staining, Expressing, Construct, Western Blot, Immunoprecipitation, Control, Generated, Mass Spectrometry