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ubash3a protein  (Proteintech)


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    Proteintech ubash3a protein
    Intracellular portion of FCRL3 interacts with <t>UBASH3A.</t> (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: .
    Ubash3a Protein, supplied by Proteintech, used in various techniques. Bioz Stars score: 94/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/ubash3a+protein/UBASH3A-Specific+Antibody/pmc12524113-324-1-15
    Average 94 stars, based on 7 article reviews
    ubash3a protein - by Bioz Stars, 2026-09
    94/100 stars

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    1) Product Images from "FCRL3 is an immunoregulatory receptor that restrains the activation of human memory T lymphocytes"

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

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20242474

    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: .
    Figure Legend 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: .

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

    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: .
    Figure Legend 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: .

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

    Related Articles

    Expressing:

    Article Title: FCRL3 is an immunoregulatory receptor that restrains the activation of human memory T lymphocytes
    Article Snippet: A similar protocol was used for UBASH3A proximity labeling, except that after washing once with wash buffer, the cells were fixed with reagent A (BD IntraSure Kit) for 5 min at room temperature, before permeabilization in digitonin buffer.A similar protocol was used for UBASH3A proximity labeling, except that after washing once with wash buffer, the cells were fixed with reagent A (BD IntraSure Kit) for 5 min at room temperature, before permeabilization in digitonin buffer.. The UBASH3A protein was targeted by incubating the cells with 4 μg of UBASH3A antibody (Proteintech) for 35 min in digitonin buffer.. After removal of the unbound antibody with two washes in digitonin buffer, cells were pelleted at 500 × g for 15 min at room temperature and incubated with digitonin buffer containing 4 μg of Protein A-TurboID ligase per sample for 45 min at 4°C using a rotator.After removal of the unbound antibody with two washes in digitonin buffer, cells were pelleted at 500 × g for 15 min at room temperature and incubated with digitonin buffer containing 4 μg of Protein A-TurboID ligase per sample for 45 min at 4°C using a rotator.

    Mass Spectrometry:

    Article Title: FCRL3 is an immunoregulatory receptor that restrains the activation of human memory T lymphocytes
    Article Snippet: A similar protocol was used for UBASH3A proximity labeling, except that after washing once with wash buffer, the cells were fixed with reagent A (BD IntraSure Kit) for 5 min at room temperature, before permeabilization in digitonin buffer.A similar protocol was used for UBASH3A proximity labeling, except that after washing once with wash buffer, the cells were fixed with reagent A (BD IntraSure Kit) for 5 min at room temperature, before permeabilization in digitonin buffer.. The UBASH3A protein was targeted by incubating the cells with 4 μg of UBASH3A antibody (Proteintech) for 35 min in digitonin buffer.. After removal of the unbound antibody with two washes in digitonin buffer, cells were pelleted at 500 × g for 15 min at room temperature and incubated with digitonin buffer containing 4 μg of Protein A-TurboID ligase per sample for 45 min at 4°C using a rotator.After removal of the unbound antibody with two washes in digitonin buffer, cells were pelleted at 500 × g for 15 min at room temperature and incubated with digitonin buffer containing 4 μg of Protein A-TurboID ligase per sample for 45 min at 4°C using a rotator.

    Incubation:

    Article Title: FCRL3 is an immunoregulatory receptor that restrains the activation of human memory T lymphocytes
    Article Snippet: A similar protocol was used for UBASH3A proximity labeling, except that after washing once with wash buffer, the cells were fixed with reagent A (BD IntraSure Kit) for 5 min at room temperature, before permeabilization in digitonin buffer.A similar protocol was used for UBASH3A proximity labeling, except that after washing once with wash buffer, the cells were fixed with reagent A (BD IntraSure Kit) for 5 min at room temperature, before permeabilization in digitonin buffer.. The UBASH3A protein was targeted by incubating the cells with 4 μg of UBASH3A antibody (Proteintech) for 35 min in digitonin buffer.. After removal of the unbound antibody with two washes in digitonin buffer, cells were pelleted at 500 × g for 15 min at room temperature and incubated with digitonin buffer containing 4 μg of Protein A-TurboID ligase per sample for 45 min at 4°C using a rotator.After removal of the unbound antibody with two washes in digitonin buffer, cells were pelleted at 500 × g for 15 min at room temperature and incubated with digitonin buffer containing 4 μg of Protein A-TurboID ligase per sample for 45 min at 4°C using a rotator.

    Plasmid Preparation:

    Article Title: FCRL3 is an immunoregulatory receptor that restrains the activation of human memory T lymphocytes
    Article Snippet: A similar protocol was used for UBASH3A proximity labeling, except that after washing once with wash buffer, the cells were fixed with reagent A (BD IntraSure Kit) for 5 min at room temperature, before permeabilization in digitonin buffer.A similar protocol was used for UBASH3A proximity labeling, except that after washing once with wash buffer, the cells were fixed with reagent A (BD IntraSure Kit) for 5 min at room temperature, before permeabilization in digitonin buffer.. The UBASH3A protein was targeted by incubating the cells with 4 μg of UBASH3A antibody (Proteintech) for 35 min in digitonin buffer.. After removal of the unbound antibody with two washes in digitonin buffer, cells were pelleted at 500 × g for 15 min at room temperature and incubated with digitonin buffer containing 4 μg of Protein A-TurboID ligase per sample for 45 min at 4°C using a rotator.After removal of the unbound antibody with two washes in digitonin buffer, cells were pelleted at 500 × g for 15 min at room temperature and incubated with digitonin buffer containing 4 μg of Protein A-TurboID ligase per sample for 45 min at 4°C using a rotator.

    Control:

    Article Title: FCRL3 is an immunoregulatory receptor that restrains the activation of human memory T lymphocytes
    Article Snippet: A similar protocol was used for UBASH3A proximity labeling, except that after washing once with wash buffer, the cells were fixed with reagent A (BD IntraSure Kit) for 5 min at room temperature, before permeabilization in digitonin buffer.A similar protocol was used for UBASH3A proximity labeling, except that after washing once with wash buffer, the cells were fixed with reagent A (BD IntraSure Kit) for 5 min at room temperature, before permeabilization in digitonin buffer.. The UBASH3A protein was targeted by incubating the cells with 4 μg of UBASH3A antibody (Proteintech) for 35 min in digitonin buffer.. After removal of the unbound antibody with two washes in digitonin buffer, cells were pelleted at 500 × g for 15 min at room temperature and incubated with digitonin buffer containing 4 μg of Protein A-TurboID ligase per sample for 45 min at 4°C using a rotator.After removal of the unbound antibody with two washes in digitonin buffer, cells were pelleted at 500 × g for 15 min at room temperature and incubated with digitonin buffer containing 4 μg of Protein A-TurboID ligase per sample for 45 min at 4°C using a rotator.

    Recombinant:

    Article Title: FCRL3 is an immunoregulatory receptor that restrains the activation of human memory T lymphocytes
    Article Snippet: A similar protocol was used for UBASH3A proximity labeling, except that after washing once with wash buffer, the cells were fixed with reagent A (BD IntraSure Kit) for 5 min at room temperature, before permeabilization in digitonin buffer.A similar protocol was used for UBASH3A proximity labeling, except that after washing once with wash buffer, the cells were fixed with reagent A (BD IntraSure Kit) for 5 min at room temperature, before permeabilization in digitonin buffer.. The UBASH3A protein was targeted by incubating the cells with 4 μg of UBASH3A antibody (Proteintech) for 35 min in digitonin buffer.. After removal of the unbound antibody with two washes in digitonin buffer, cells were pelleted at 500 × g for 15 min at room temperature and incubated with digitonin buffer containing 4 μg of Protein A-TurboID ligase per sample for 45 min at 4°C using a rotator.After removal of the unbound antibody with two washes in digitonin buffer, cells were pelleted at 500 × g for 15 min at room temperature and incubated with digitonin buffer containing 4 μg of Protein A-TurboID ligase per sample for 45 min at 4°C using a rotator.

    Western Blot:

    Article Title: FCRL3 is an immunoregulatory receptor that restrains the activation of human memory T lymphocytes
    Article Snippet: A similar protocol was used for UBASH3A proximity labeling, except that after washing once with wash buffer, the cells were fixed with reagent A (BD IntraSure Kit) for 5 min at room temperature, before permeabilization in digitonin buffer.A similar protocol was used for UBASH3A proximity labeling, except that after washing once with wash buffer, the cells were fixed with reagent A (BD IntraSure Kit) for 5 min at room temperature, before permeabilization in digitonin buffer.. The UBASH3A protein was targeted by incubating the cells with 4 μg of UBASH3A antibody (Proteintech) for 35 min in digitonin buffer.. After removal of the unbound antibody with two washes in digitonin buffer, cells were pelleted at 500 × g for 15 min at room temperature and incubated with digitonin buffer containing 4 μg of Protein A-TurboID ligase per sample for 45 min at 4°C using a rotator.After removal of the unbound antibody with two washes in digitonin buffer, cells were pelleted at 500 × g for 15 min at room temperature and incubated with digitonin buffer containing 4 μg of Protein A-TurboID ligase per sample for 45 min at 4°C using a rotator.

    Protein-Protein interactions:

    Article Title: FCRL3 is an immunoregulatory receptor that restrains the activation of human memory T lymphocytes
    Article Snippet: A similar protocol was used for UBASH3A proximity labeling, except that after washing once with wash buffer, the cells were fixed with reagent A (BD IntraSure Kit) for 5 min at room temperature, before permeabilization in digitonin buffer.A similar protocol was used for UBASH3A proximity labeling, except that after washing once with wash buffer, the cells were fixed with reagent A (BD IntraSure Kit) for 5 min at room temperature, before permeabilization in digitonin buffer.. The UBASH3A protein was targeted by incubating the cells with 4 μg of UBASH3A antibody (Proteintech) for 35 min in digitonin buffer.. After removal of the unbound antibody with two washes in digitonin buffer, cells were pelleted at 500 × g for 15 min at room temperature and incubated with digitonin buffer containing 4 μg of Protein A-TurboID ligase per sample for 45 min at 4°C using a rotator.After removal of the unbound antibody with two washes in digitonin buffer, cells were pelleted at 500 × g for 15 min at room temperature and incubated with digitonin buffer containing 4 μg of Protein A-TurboID ligase per sample for 45 min at 4°C using a rotator.

    Co-Immunoprecipitation Assay:

    Article Title: FCRL3 is an immunoregulatory receptor that restrains the activation of human memory T lymphocytes
    Article Snippet: A similar protocol was used for UBASH3A proximity labeling, except that after washing once with wash buffer, the cells were fixed with reagent A (BD IntraSure Kit) for 5 min at room temperature, before permeabilization in digitonin buffer.A similar protocol was used for UBASH3A proximity labeling, except that after washing once with wash buffer, the cells were fixed with reagent A (BD IntraSure Kit) for 5 min at room temperature, before permeabilization in digitonin buffer.. The UBASH3A protein was targeted by incubating the cells with 4 μg of UBASH3A antibody (Proteintech) for 35 min in digitonin buffer.. After removal of the unbound antibody with two washes in digitonin buffer, cells were pelleted at 500 × g for 15 min at room temperature and incubated with digitonin buffer containing 4 μg of Protein A-TurboID ligase per sample for 45 min at 4°C using a rotator.After removal of the unbound antibody with two washes in digitonin buffer, cells were pelleted at 500 × g for 15 min at room temperature and incubated with digitonin buffer containing 4 μg of Protein A-TurboID ligase per sample for 45 min at 4°C using a rotator.

    Transfection:

    Article Title: FCRL3 is an immunoregulatory receptor that restrains the activation of human memory T lymphocytes
    Article Snippet: A similar protocol was used for UBASH3A proximity labeling, except that after washing once with wash buffer, the cells were fixed with reagent A (BD IntraSure Kit) for 5 min at room temperature, before permeabilization in digitonin buffer.A similar protocol was used for UBASH3A proximity labeling, except that after washing once with wash buffer, the cells were fixed with reagent A (BD IntraSure Kit) for 5 min at room temperature, before permeabilization in digitonin buffer.. The UBASH3A protein was targeted by incubating the cells with 4 μg of UBASH3A antibody (Proteintech) for 35 min in digitonin buffer.. After removal of the unbound antibody with two washes in digitonin buffer, cells were pelleted at 500 × g for 15 min at room temperature and incubated with digitonin buffer containing 4 μg of Protein A-TurboID ligase per sample for 45 min at 4°C using a rotator.After removal of the unbound antibody with two washes in digitonin buffer, cells were pelleted at 500 × g for 15 min at room temperature and incubated with digitonin buffer containing 4 μg of Protein A-TurboID ligase per sample for 45 min at 4°C using a rotator.

    Transduction:

    Article Title: FCRL3 is an immunoregulatory receptor that restrains the activation of human memory T lymphocytes
    Article Snippet: A similar protocol was used for UBASH3A proximity labeling, except that after washing once with wash buffer, the cells were fixed with reagent A (BD IntraSure Kit) for 5 min at room temperature, before permeabilization in digitonin buffer.A similar protocol was used for UBASH3A proximity labeling, except that after washing once with wash buffer, the cells were fixed with reagent A (BD IntraSure Kit) for 5 min at room temperature, before permeabilization in digitonin buffer.. The UBASH3A protein was targeted by incubating the cells with 4 μg of UBASH3A antibody (Proteintech) for 35 min in digitonin buffer.. After removal of the unbound antibody with two washes in digitonin buffer, cells were pelleted at 500 × g for 15 min at room temperature and incubated with digitonin buffer containing 4 μg of Protein A-TurboID ligase per sample for 45 min at 4°C using a rotator.After removal of the unbound antibody with two washes in digitonin buffer, cells were pelleted at 500 × g for 15 min at room temperature and incubated with digitonin buffer containing 4 μg of Protein A-TurboID ligase per sample for 45 min at 4°C using a rotator.

    Immunoprecipitation:

    Article Title: FCRL3 is an immunoregulatory receptor that restrains the activation of human memory T lymphocytes
    Article Snippet: A similar protocol was used for UBASH3A proximity labeling, except that after washing once with wash buffer, the cells were fixed with reagent A (BD IntraSure Kit) for 5 min at room temperature, before permeabilization in digitonin buffer.A similar protocol was used for UBASH3A proximity labeling, except that after washing once with wash buffer, the cells were fixed with reagent A (BD IntraSure Kit) for 5 min at room temperature, before permeabilization in digitonin buffer.. The UBASH3A protein was targeted by incubating the cells with 4 μg of UBASH3A antibody (Proteintech) for 35 min in digitonin buffer.. After removal of the unbound antibody with two washes in digitonin buffer, cells were pelleted at 500 × g for 15 min at room temperature and incubated with digitonin buffer containing 4 μg of Protein A-TurboID ligase per sample for 45 min at 4°C using a rotator.After removal of the unbound antibody with two washes in digitonin buffer, cells were pelleted at 500 × g for 15 min at room temperature and incubated with digitonin buffer containing 4 μg of Protein A-TurboID ligase per sample for 45 min at 4°C using a rotator.

    Staining:

    Article Title: FCRL3 is an immunoregulatory receptor that restrains the activation of human memory T lymphocytes
    Article Snippet: A similar protocol was used for UBASH3A proximity labeling, except that after washing once with wash buffer, the cells were fixed with reagent A (BD IntraSure Kit) for 5 min at room temperature, before permeabilization in digitonin buffer.A similar protocol was used for UBASH3A proximity labeling, except that after washing once with wash buffer, the cells were fixed with reagent A (BD IntraSure Kit) for 5 min at room temperature, before permeabilization in digitonin buffer.. The UBASH3A protein was targeted by incubating the cells with 4 μg of UBASH3A antibody (Proteintech) for 35 min in digitonin buffer.. After removal of the unbound antibody with two washes in digitonin buffer, cells were pelleted at 500 × g for 15 min at room temperature and incubated with digitonin buffer containing 4 μg of Protein A-TurboID ligase per sample for 45 min at 4°C using a rotator.After removal of the unbound antibody with two washes in digitonin buffer, cells were pelleted at 500 × g for 15 min at room temperature and incubated with digitonin buffer containing 4 μg of Protein A-TurboID ligase per sample for 45 min at 4°C using a rotator.

    Construct:

    Article Title: FCRL3 is an immunoregulatory receptor that restrains the activation of human memory T lymphocytes
    Article Snippet: A similar protocol was used for UBASH3A proximity labeling, except that after washing once with wash buffer, the cells were fixed with reagent A (BD IntraSure Kit) for 5 min at room temperature, before permeabilization in digitonin buffer.A similar protocol was used for UBASH3A proximity labeling, except that after washing once with wash buffer, the cells were fixed with reagent A (BD IntraSure Kit) for 5 min at room temperature, before permeabilization in digitonin buffer.. The UBASH3A protein was targeted by incubating the cells with 4 μg of UBASH3A antibody (Proteintech) for 35 min in digitonin buffer.. After removal of the unbound antibody with two washes in digitonin buffer, cells were pelleted at 500 × g for 15 min at room temperature and incubated with digitonin buffer containing 4 μg of Protein A-TurboID ligase per sample for 45 min at 4°C using a rotator.After removal of the unbound antibody with two washes in digitonin buffer, cells were pelleted at 500 × g for 15 min at room temperature and incubated with digitonin buffer containing 4 μg of Protein A-TurboID ligase per sample for 45 min at 4°C using a rotator.

    Generated:

    Article Title: FCRL3 is an immunoregulatory receptor that restrains the activation of human memory T lymphocytes
    Article Snippet: A similar protocol was used for UBASH3A proximity labeling, except that after washing once with wash buffer, the cells were fixed with reagent A (BD IntraSure Kit) for 5 min at room temperature, before permeabilization in digitonin buffer.A similar protocol was used for UBASH3A proximity labeling, except that after washing once with wash buffer, the cells were fixed with reagent A (BD IntraSure Kit) for 5 min at room temperature, before permeabilization in digitonin buffer.. The UBASH3A protein was targeted by incubating the cells with 4 μg of UBASH3A antibody (Proteintech) for 35 min in digitonin buffer.. After removal of the unbound antibody with two washes in digitonin buffer, cells were pelleted at 500 × g for 15 min at room temperature and incubated with digitonin buffer containing 4 μg of Protein A-TurboID ligase per sample for 45 min at 4°C using a rotator.After removal of the unbound antibody with two washes in digitonin buffer, cells were pelleted at 500 × g for 15 min at room temperature and incubated with digitonin buffer containing 4 μg of Protein A-TurboID ligase per sample for 45 min at 4°C using a rotator.



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    94
    Proteintech ubash3a protein
    Intracellular portion of FCRL3 interacts with <t>UBASH3A.</t> (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: .
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    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: .

    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

    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: .

    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