Figure 6.
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 (log2FC ≥ |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 (log2FC ≥ |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 Data S4. Source data are available for this figure: SourceData F6.

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 (log2FC ≥ |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 (log2FC ≥ |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 Data S4. Source data are available for this figure: SourceData F6.

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