anti human plcγ1 antibody  (Cell Signaling Technology Inc)

Journal: The Journal of Cell Biology

Article Title: PLCγ1 promotes phase separation of T cell signaling components

doi: 10.1083/jcb.202009154

Figure Lengend Snippet: LAT cluster formation with Grb2, Sos1, and PLCγ1. Same condition as in Video 1 except that PLCγ1 (50 nM) was added together with Grb2 and Sos1 at 0 s. The video was recorded at a rate of 10 s/frame.

Article Snippet: Antibody , Anti-human PLCγ1 pY783 , Cell Signaling , Cat#2821, Western blot (1:3,000).

Techniques:

Journal: The Journal of Cell Biology

Article Title: PLCγ1 promotes phase separation of T cell signaling components

doi: 10.1083/jcb.202009154

Figure Lengend Snippet: Early phase of simulation of LAT cluster formation at low PLCγ1-to-LAT ratio. The simulation involves 200 LAT, 15 PLCγ1, 400 Grb2, and 200 Sos1 molecules, all in a monomeric state. The simulation starts at second 2.00, corresponding to timestep 0, and ends at timestep 10 × 10 6 . The interval between two frames is 0.2 × 10 6 timesteps, and the frame rate is 8 s −1 . Particle scheme is the same as in Fig. 6 A : gray, LAT; yellow, PLCγ1; blue, Grb2; and pink, Sos1.

Article Snippet: Antibody , Anti-human PLCγ1 pY783 , Cell Signaling , Cat#2821, Western blot (1:3,000).

Techniques:

Journal: The Journal of Cell Biology

Article Title: PLCγ1 promotes phase separation of T cell signaling components

doi: 10.1083/jcb.202009154

Figure Lengend Snippet: Full-length simulation of LAT cluster formation at low PLCγ1-to-LAT ratio. High-resolution video for visualizing individual chemical bonds. The simulation involves 200 LAT, 15 PLCγ1, 400 Grb2, and 200 Sos1 molecules, all in a monomeric state. The simulation starts at second 2.00, corresponding to timestep 0, and ends at timestep 50 × 10 6 . The interval between two frames is 0.2 × 10 6 timesteps, and the frame rate is 16 s −1 . Particle scheme is the same as in Fig. 6 A : gray, LAT; yellow, PLCγ1; blue, Grb2; and pink, Sos1.

Article Snippet: Antibody , Anti-human PLCγ1 pY783 , Cell Signaling , Cat#2821, Western blot (1:3,000).

Techniques:

Journal: The Journal of Cell Biology

Article Title: PLCγ1 promotes phase separation of T cell signaling components

doi: 10.1083/jcb.202009154

Figure Lengend Snippet: Early phase of simulation of LAT cluster formation at intermediate PLCγ1-to-LAT ratio. The simulation involves 200 LAT, 150 PLCγ1, 400 Grb2, and 200 Sos1 molecules, all in a monomeric state. The simulation starts at second 2.00, corresponding to timestep 0, and ends at timestep 10 × 10 6 . The interval between two frames is 0.2 × 10 6 timesteps, and the frame rate is 8 s −1 . Particle scheme is the same as in Fig. 6 A : gray, LAT; yellow, PLCγ1; blue, Grb2; and pink, Sos1.

Article Snippet: Antibody , Anti-human PLCγ1 pY783 , Cell Signaling , Cat#2821, Western blot (1:3,000).

Techniques:

Journal: The Journal of Cell Biology

Article Title: PLCγ1 promotes phase separation of T cell signaling components

doi: 10.1083/jcb.202009154

Figure Lengend Snippet: Full-length simulation of LAT cluster formation at intermediate PLCγ1-to-LAT ratio. High-resolution video for visualizing individual chemical bonds. The simulation involves 200 LAT, 150 PLCγ1, 400 Grb2, and 200 Sos1 molecules, all in a monomeric state. The simulation starts at second 2.00, corresponding to timestep 0, and ends at timestep 50 × 10 6 . The interval between two frames is 0.2 × 10 6 timesteps, and the frame rate is 16 s −1 . Particle scheme is the same as in Fig. 6 A : gray, LAT; yellow, PLCγ1; blue, Grb2; and pink, Sos1.

Article Snippet: Antibody , Anti-human PLCγ1 pY783 , Cell Signaling , Cat#2821, Western blot (1:3,000).

Techniques:

Journal: The Journal of Cell Biology

Article Title: PLCγ1 promotes phase separation of T cell signaling components

doi: 10.1083/jcb.202009154

Figure Lengend Snippet: Early phase of simulation of LAT cluster formation at high PLCγ1-to-LAT ratio. The simulation involves 200 LAT, 600 PLCγ1, 400 Grb2, and 200 Sos1 molecules, all in a monomeric state. The simulation starts at second 2.00, corresponding to timestep 0, and ends at timestep 10 × 10 6 . The interval between two frames is 0.2 × 10 6 timesteps, and the frame rate is 8 s −1 . Particle scheme is the same as in Fig. 6 A : gray, LAT; yellow, PLCγ1; blue, Grb2; and pink, Sos1.

Article Snippet: Antibody , Anti-human PLCγ1 pY783 , Cell Signaling , Cat#2821, Western blot (1:3,000).

Techniques:

Journal: The Journal of Cell Biology

Article Title: PLCγ1 promotes phase separation of T cell signaling components

doi: 10.1083/jcb.202009154

Figure Lengend Snippet: Full-length simulation of LAT cluster formation at high PLCγ1-to-LAT ratio. High-resolution video for visualizing individual chemical bonds. The simulation involves 200 LAT, 600 PLCγ1, 400 Grb2, and 200 Sos1 molecules, all in a monomeric state. The simulation starts at second 2.00, corresponding to timestep 0, and ends at timestep 50 × 10 6 . The interval between two frames is 0.2 × 10 6 timesteps, and the frame rate is 16 s −1 . Particle scheme is the same as in Fig. 6 A : gray, LAT; yellow, PLCγ1; blue, Grb2; and pink, Sos1.

Article Snippet: Antibody , Anti-human PLCγ1 pY783 , Cell Signaling , Cat#2821, Western blot (1:3,000).

Techniques:

Journal: The Journal of Cell Biology

Article Title: PLCγ1 promotes phase separation of T cell signaling components

doi: 10.1083/jcb.202009154

Figure Lengend Snippet: PLCγ1 promotes LAT cluster formation in vitro. (A) Domain structure of PLCγ1. (B) Schematics of the assay. (C) Top: TIRF microscopy revealed that both Grb2 and PLCγ1 (full-length) promote LAT microcluster formation. Alexa Fluor 488–labeled LAT at 300 molecules/µm 2 was incubated with 125 nM Sos1 and 250 nM Grb2 or PLCγ1 for 0.5 h before imaging. Scale bar, 5 µm. Bottom: Quantification of Grb2- or PLCγ1-driven LAT microclusters. LAT clustering was quantified as normalized variance . Shown are mean ± SD; n = 3 independent experiments. Unpaired two-tailed t test was used. *, P < 0.05; **, P < 0.01. (D) FRAP analysis revealed that PLCγ1-driven microclusters are less dynamic than Grb2-driven LAT microclusters. Shown are mean ± SD; n = 10 clusters.

Article Snippet: Antibody , Anti-human PLCγ1 pY783 , Cell Signaling , Cat#2821, Western blot (1:3,000).

Techniques: In Vitro, Microscopy, Labeling, Incubation, Imaging, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: PLCγ1 promotes phase separation of T cell signaling components

doi: 10.1083/jcb.202009154

Figure Lengend Snippet: Both the full-length and SH fragment of PLCγ1 drive LAT clustering. (A) TIRF microscopy revealed LAT microcluster formation with the full-length or SH2-SH2-SH3 domain of PLCγ1. Alexa Fluor 488–labeled, phosphorylated LAT at 300 molecules/µm 2 was incubated with 250 nM Sos1 and indicated concentrations of PLCγ1 or fragment. Scale bar, 5 µm. (B) Quantification of PLCγ1-driven LAT microclusters. Shown are mean ± SD; n = 3 independent experiments. (C) Recombinant proteins used in this study. Purified proteins were applied to SDS-PAGE, followed by Coomassie blue staining.

Article Snippet: Antibody , Anti-human PLCγ1 pY783 , Cell Signaling , Cat#2821, Western blot (1:3,000).

Techniques: Microscopy, Labeling, Incubation, Recombinant, Purification, SDS Page, Staining

Journal: The Journal of Cell Biology

Article Title: PLCγ1 promotes phase separation of T cell signaling components

doi: 10.1083/jcb.202009154

Figure Lengend Snippet: PLCγ1 cross-links LAT by two SH2 domains. (A) Domains of the proteins used in the study. (B) TIRF microscopy revealed that both nSH2 and cSH2 are required for PLCγ1-driven LAT microcluster formation. SH3 domain promotes cluster formation. Alexa Fluor 488–labeled LAT at 300 molecules/µm 2 was incubated with 300 nM Sos1 and 50 nM PLCγ1 for 0.5 h before imaging. Scale bar, 5 µm. (C) Quantification of PLCγ1-driven LAT microclusters. Shown are mean ± SD; n = 3 independent experiments. Unpaired two-tailed t test was used. *, P < 0.05. (D) Schematics of the assay of testing SH2 domain binding sites. (E) PLCγ1 nSH2 binds LAT Y132. Phosphopeptides were synthesized, biotinylated at the N terminus, and attached to the biotin-functionalized supported lipid bilayers by streptavidin. The SH2 domains were labeled with fluorescent dye (Maleimide-Ax647) and incubated with the individual phosphopeptides. The membrane-associated SH2 domain was measured by TIRF microscopy. Scale bar, 5 µm. (F) PLCγ1 cSH2 binds LAT Y171. Same settings as in E.

Article Snippet: Antibody , Anti-human PLCγ1 pY783 , Cell Signaling , Cat#2821, Western blot (1:3,000).

Techniques: Microscopy, Labeling, Incubation, Imaging, Two Tailed Test, Binding Assay, Synthesized, Membrane

Journal: The Journal of Cell Biology

Article Title: PLCγ1 promotes phase separation of T cell signaling components

doi: 10.1083/jcb.202009154

Figure Lengend Snippet: Domains required for PLCγ1-driven LAT clustering. (A) TIRF microscopy revealed LAT microcluster formation with a high concentration of PLCγ1 fragments. Alexa Fluor 488 LAT at 300 molecules/µm 2 was incubated with 125 nM Sos1 and 500 nM of indicated PLCγ1 fragments. Scale bar, 5 µm. (B) Quantification of LAT clustering in A. Shown are mean ± SD; n = 3 independent experiments. (C) TIRF microscopy revealed LAT microcluster formation in the presence or absence of PLCγ1. Alexa Fluor 488 LAT at 1,000 molecules/µm 2 was incubated with 500 nM Sos1 and 1,000 nM Grb2 with or without 100 nM full-length PLCγ1. Scale bar, 5 µm. (D) Quantification of LAT clustering. Shown are mean ± SD; n = 3 independent experiments. Unpaired two-tailed t test. ***, P < 0.001. (E) FRAP analysis revealed that PLCγ1 decreases the recovery of LAT signal in clusters after photobleaching. Shown are mean ± SD; n = 10 clusters.

Article Snippet: Antibody , Anti-human PLCγ1 pY783 , Cell Signaling , Cat#2821, Western blot (1:3,000).

Techniques: Microscopy, Concentration Assay, Incubation, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: PLCγ1 promotes phase separation of T cell signaling components

doi: 10.1083/jcb.202009154

Figure Lengend Snippet: Sos1 facilitates PLCγ1-driven LAT clustering. (A) TIRF microscopy revealed LAT microcluster formation with titrated PLCγ1. Alexa Fluor 488 LAT at 300 molecules/µm 2 was incubated with or without 250 nM Sos1 and the indicated concentration of PLCγ1 nSH2-cSH2-SH3 domains. Scale bar, 5 µm. (B) Quantification of LAT clustering in A. Shown are mean ± SD; n = 3 independent experiments. Unpaired two-tailed t test. *, P < 0.05; **, P < 0.01. (C) Binding affinity (Kd) of the SH3 domain of PLCγ1 to the PRM on Sos1 as measured by surface plasmon resonance. Shown are mean ± SEM. (D) TIRF microscopy revealed that SLP76 inhibits LAT microcluster formation driven by PLCγ1 and Sos1. Alexa Fluor 488 LAT at 300 molecules/µm 2 was incubated with 300 nM Sos1, 50 nM PLCγ1 (nSH2-cSH2-SH3 domains), and the indicated concentration of SLP76. Proteins were used at the physiologically relevant concentration. The cellular concentration of SLP76 was estimated as 150 nM (by MaxQB Database). Scale bar, 5 µm. (E) Quantification of LAT clustering in D. Shown are mean ± SD; n = 3 independent experiments. Unpaired two-tailed t test. *, P < 0.05; ns, not significant.

Article Snippet: Antibody , Anti-human PLCγ1 pY783 , Cell Signaling , Cat#2821, Western blot (1:3,000).

Techniques: Microscopy, Incubation, Concentration Assay, Two Tailed Test, Binding Assay, SPR Assay

Journal: The Journal of Cell Biology

Article Title: PLCγ1 promotes phase separation of T cell signaling components

doi: 10.1083/jcb.202009154

Figure Lengend Snippet: PLCγ1 cooperates with Grb2 to regulate LAT microcluster formation. (A) TIRF microscopy revealed that PLCγ1 regulates LAT microcluster formation in a nonmonotonic manner. Physiologically relevant concentrations of proteins were used in the assay: LAT at 300 molecules/µm 2 , Grb2 at 3 µM, Sos1 at 0.3 µM, and PLCγ1 at 50 nM. LAT was labeled with Alexa Fluor 488, PLCγ1 (SH2-2-3 domains) was labeled with DY547, and Sos1 was labeled with Alexa Fluor 647. Scale bar, 5 µm. (B) Quantification of LAT clustering, membrane recruitment of Sos1. Shown are mean ± SD; n = 3 independent experiments. Unpaired two-tailed t test was used. **, P < 0.01. (C) PLCγ1 accelerates LAT cluster formation. TIRF microscopy revealed the time course of LAT microcluster formation in the presence or absence of PLCγ1. LAT–Alexa Fluor 488 at 1,000 molecules/µm 2 was incubated with 1,000 nM Grb2 and 500 nM Sos1 and/or 50 nM PLCγ1 at time 0. Shown are mean ± SEM; n = 3 independent experiments.

Article Snippet: Antibody , Anti-human PLCγ1 pY783 , Cell Signaling , Cat#2821, Western blot (1:3,000).

Techniques: Microscopy, Labeling, Membrane, Two Tailed Test, Incubation

Journal: The Journal of Cell Biology

Article Title: PLCγ1 promotes phase separation of T cell signaling components

doi: 10.1083/jcb.202009154

Figure Lengend Snippet: A coarse-grained model explains how PLCγ1 nonmonotonically regulates LAT clustering. (A) Sketch of the model in which the proteins are represented as 2D particles decorated by interaction patches. All bonds possible in the system, based on biochemical data, are illustrated with colored lines. (B) Top: The average cluster size displays nonmonotonic dependence on the PLCγ1 concentration (gray circles). This behavior is well captured by the likelihood for cluster coalescence (black squares). Error bars represent statistical errors on the average size over 10 different realizations of the simulation, shown are mean ± SEM. Bottom: Snapshots of typical clusters in simulations, for relative PLCγ1:LAT concentrations of 0.075, 0.75, and 3 (these clusters contain, respectively, 19, 30, and 10 LAT molecules, and with reference to D, their compactness is 0.23, 0.39, and 0.48). (C) Breakdown of the coalescence likelihood per type of possible bond. The gray and pink areas represent available bonds involving a LAT or a Sos1 molecule, respectively; blue and yellow-orange bars represent bonds involving Grb2 and PLCγ1, respectively. (D) Compactness (gray circles, see Materials and methods) and fraction of terminal nodes (black squares), as a function of PLCγ1 concentration. Shown are mean ± SD; n = 10 realizations. (E) Fraction of LAT, PLCγ1, Sos1, and Grb2 molecules per cluster, as a function of PLCγ1 concentration, shaded according to the number of other molecules they are bound to.

Article Snippet: Antibody , Anti-human PLCγ1 pY783 , Cell Signaling , Cat#2821, Western blot (1:3,000).

Techniques: Concentration Assay

Journal: The Journal of Cell Biology

Article Title: PLCγ1 promotes phase separation of T cell signaling components

doi: 10.1083/jcb.202009154

Figure Lengend Snippet: Simulating PLCγ1’s effect on LAT cluster size and bond type. (A) The effect of PLCγ1 on LAT clustering is independent of LAT density ρ LAT . Coarse-grained model simulating LAT clustering as a function of PLCγ1 concentration. In a wide range of LAT densities tested, PLCγ1 regulates LAT clustering in a nonmonotonic manner. LAT clusters are quantified by the number of LAT in each cluster (solid line) or the total number of molecules (LAT, Grb2, PLCγ1, or Sos1) in each cluster (dashed line, scaled down by a factor of 4 to fit in the same plot). Surface densities ρ LAT are in units of σ −2 , where σ is the diameter of a particle, of the order of a few nanometers, and our experiments correspond roughly to ρLAT = 0.02σ −2 (see Materials and methods and Simulation details). Shown are mean ± SEM. (B) Average coordination number for all four kinds of particles, as a function of ratio of PLCγ1:LAT, broken down to the contribution of each specific bond. Yellow-orange bars represent bonds involving PLCγ1, and blue bars, Grb2; a gray background represents bonds involving LAT, and a pink background, Sos1. Here, as throughout , ρ LAT = 0.02 σ −2 . See , section SI 3, for a complete analysis. Shown are mean ± SEM.

Article Snippet: Antibody , Anti-human PLCγ1 pY783 , Cell Signaling , Cat#2821, Western blot (1:3,000).

Techniques: Concentration Assay

Journal: The Journal of Cell Biology

Article Title: PLCγ1 promotes phase separation of T cell signaling components

doi: 10.1083/jcb.202009154

Figure Lengend Snippet: PLCγ1 promotes LAT clustering, SLP76 phosphorylation, and ERK activation in Jurkat T cells. (A) Diminished LAT microcluster formation in PLCγ1-null cells. Wild-type or PLCγ1-null Jurkat T cells expressing LAT-mCherry were plated on OKT3-coated cover glass. LAT microcluster formation was revealed by TIRF microscopy. Images showed clustering 90 s after cell landing on the glass. Scale bar, 5 µm. Shown are mean ± SEM; n = 25 or 26 cells. Unpaired two-tailed t test was used. **, P < 0.01. (B) The nSH2 and SH3 domain of PLCγ1 promotes LAT cluster formation. PLCγ1-null Jurkat T cells expressing LAT-mCherry were reconstituted with the GFP-tagged wild-type, Δ nSH2, or Δ SH3 PLCγ1. Those cells were plated on OKT3-coated cover glass. Images showed clustering 90 s after cell landing on the glass. LAT microcluster formation was revealed by TIRF microscopy. Scale bar, 5 µm. Shown are mean ± SEM; n = 22–30 cells. Unpaired two-tailed t test was used. **, P < 0.01. (C) Immunoblot analysis of LAT-null Jurkat T cells reconstituted with the GFP-tagged wild-type, Δ nSH2, or Δ SH3 PLCγ1. Cells were stimulated with 2 µg/ml anti-CD3 and anti-CD28 antibodies for 2 min, lysed, and applied for Western blot analysis. MW, molecular weight. (D) Quantification of the level of indicated proteins, after being normalized to the expression level of GAPDH. Shown are mean ± SD; n = 3 independent experiments. Unpaired two-tailed t test was used. *, P < 0.05; **, P < 0.01.

Article Snippet: Antibody , Anti-human PLCγ1 pY783 , Cell Signaling , Cat#2821, Western blot (1:3,000).

Techniques: Phospho-proteomics, Activation Assay, Expressing, Microscopy, Two Tailed Test, Western Blot, Molecular Weight

Journal: The Journal of Cell Biology

Article Title: PLCγ1 promotes phase separation of T cell signaling components

doi: 10.1083/jcb.202009154

Figure Lengend Snippet: The nSH2–pY interaction is required for PLCγ1-mediated LAT clustering and signaling. (A) Diminished LAT microcluster formation in cells expressing PLCγ1 R586K. R586K abolishes the nSH2 interaction with LAT pY132. PLCγ1-null Jurkat T cells that express LAT-mCherry were reconstituted with the GFP-tagged wild type or R586K PLCγ1. They were plated on OKT3-coated cover glass. LAT microcluster formation was revealed by TIRF microscopy. Left: Images showed clustering 60 s after cell landing on the glass. Scale bar, 5 µm. Right: Quantification of clustering. Shown are mean ± SEM; n = 23–29 cells. Unpaired two-tailed t test was used. *, P < 0.05. (B) Immunoblot analysis of LAT-null Jurkat T cells reconstituted with the GFP-tagged wild type or R586K PLCγ1. Cells were stimulated with 2 µg/ml anti-CD3 and anti-CD28 antibodies for 2 min, lysed, and applied for Western blot analysis. MW, molecular weight. (C) Quantification of the level of indicated proteins, after being normalized to the expression level of GAPDH. Shown are mean ± SD; n = 3 independent experiments. Unpaired two-tailed t test was used. *, P < 0.05; ***, P < 0.001.

Article Snippet: Antibody , Anti-human PLCγ1 pY783 , Cell Signaling , Cat#2821, Western blot (1:3,000).

Techniques: Expressing, Microscopy, Two Tailed Test, Western Blot, Molecular Weight

Journal: The Journal of Cell Biology

Article Title: PLCγ1 promotes phase separation of T cell signaling components

doi: 10.1083/jcb.202009154

Figure Lengend Snippet: The lipase-independent signaling role of PLCγ1. (A) Immunoblot analysis of LAT-null Jurkat T cells reconstituted with the GFP-tagged wild type or H380F PLCγ1. H380F abolishes most of the enzymatic activity of PLCγ1. Cells were stimulated with 2 µg/ml anti-CD3 and anti-CD28 antibodies for 2 min, lysed, and applied for Western blot analysis. MW, molecular weight. (B) Quantification of the level of indicated proteins, after being normalized to the expression level of GAPDH. Shown are mean ± SD; n = 3 independent experiments. Unpaired two-tailed t test was used. **, P < 0.01. (C) Cells as indicated were stimulated with 2 µg/ml anti-CD3 and anti-CD28 antibodies for 2 min, lysed, and applied for Western blot analysis. (D) The level of LAT pY171, after being normalized to the level of GAPDH, was quantified. Shown are mean ± SD; n = 3 independent experiments.

Article Snippet: Antibody , Anti-human PLCγ1 pY783 , Cell Signaling , Cat#2821, Western blot (1:3,000).

Techniques: Western Blot, Activity Assay, Molecular Weight, Expressing, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: PLCγ1 promotes phase separation of T cell signaling components

doi: 10.1083/jcb.202009154

Figure Lengend Snippet: PLCγ1 protects LAT from dephosphorylation by CD45. (A) Reduced phosphorylation at LAT Y132 in PLCγ1-null cells. Cells as indicated were stimulated with 2 µg/ml anti-CD3 and anti-CD28 antibodies for 2 min, lysed, and applied for Western blot analysis. The level of indicated proteins, after being normalized to the level of GAPDH, was quantified. Shown are mean ± SD; n = 3 independent experiments. Unpaired two-tailed t test was used. **, P < 0.01. MW, molecular weight. (B) PLCγ1 prevents LAT Y132 from being dephosphorylated. Cells as indicated were pretreated with 0.1 mM vanadate (pan-phosphatase inhibitor) before being stimulated with 2 µg/ml anti-CD3 and anti-CD28 antibodies for 2 min, lysed, and applied for Western blot analysis. The level of indicated proteins, after being normalized to the level of GAPDH, was quantified. Shown are mean ± SD; n = 3 independent experiments. Unpaired two-tailed t test was used. **, P < 0.01; ns, not significant. (C) Schematics of the in intro dephosphorylation assay. (D) PLCγ1 prevents LAT Y132 from being dephosphorylated by CD45 in vitro. pLAT, at 1,000 molecules/µm 2 , was incubated with 1 µM Grb2, 0.5 µM Sos1, and/or 100 nM full-length PLCγ1 for 0.5 h. CD45 was then added to dephosphorylate pLAT for 5 min. The reaction was terminated by adding SDS-PAGE loading buffer with 2 mM vanadate. The level of phosphorylated LAT, after being normalized to total LAT, was quantified. Shown are mean ± SD; n = 3 independent experiments.

Article Snippet: Antibody , Anti-human PLCγ1 pY783 , Cell Signaling , Cat#2821, Western blot (1:3,000).

Techniques: De-Phosphorylation Assay, Phospho-proteomics, Western Blot, Two Tailed Test, Molecular Weight, In Vitro, Incubation, SDS Page

Journal: The Journal of Cell Biology

Article Title: PLCγ1 promotes phase separation of T cell signaling components

doi: 10.1083/jcb.202009154

Figure Lengend Snippet: Mechanism of phosphotyrosine protection by LAT clustering. (A) TIRF microscopy revealed clustered and unclustered LAT and Grb2. Alexa Fluor 488–labeled LAT at 1,000 molecules/µm 2 was incubated with 1 µM Grb2 + 500 nM Sos1 or 6 µM Grb2. 20% of Grb2 was labeled with Alexa Fluor 568. Similar Grb2 was recruited to the membrane in the two indicated conditions. Shown are mean ± SD; n = 3 independent experiments. Scale bar, 5 µm. (B) LAT clustering by Grb2 prevents LAT Y132 from being dephosphorylated by CD45 in vitro. pLAT, at 1,000 molecules/µm 2 , was incubated with 1 µM Grb2, 0.5 µM Sos1, or 1 µM Grb2. CD45 was added to dephosphorylate pLAT for 5 min. The reaction was terminated by adding SDS-PAGE loading buffer with 2 mM vanadate. The level of LAT pY132, after being normalized to total LAT, was quantified. Shown are mean ± SD; n = 3 independent experiments. MW, molecular weight. (C) CD45 is excluded by Grb2- or PLCγ1-mediated LAT clustering. pLAT–Alexa Fluor 488 (1,000 molecules/μm 2 ) was incubated with 1 µM Sos1 and 1 µM Grb2 or 1 µM PLCγ1-SH2-2-3 fragment. The cytoplasmic domain of CD45-TMR (4 nM, with an N-terminal His10 tag) was added, and its localization was visualized by TIRF microscopy. Scale bar, 5 µm. (D) Quantification of fluorescence intensity of pLAT and CD45 along the line scan indicated by a white line in the top merged image.

Article Snippet: Antibody , Anti-human PLCγ1 pY783 , Cell Signaling , Cat#2821, Western blot (1:3,000).

Techniques: Microscopy, Labeling, Incubation, Membrane, In Vitro, SDS Page, Molecular Weight, Fluorescence

Journal: The Journal of Cell Biology

Article Title: PLCγ1 promotes phase separation of T cell signaling components

doi: 10.1083/jcb.202009154

Figure Lengend Snippet: Key resources

Article Snippet: Antibody , Anti-human PLCγ1 pY783 , Cell Signaling , Cat#2821, Western blot (1:3,000).

Techniques: Cell Culture, Expressing, Knock-Out, CRISPR, Plasmid Preparation, Infection, Bacteria, Recombinant, Western Blot

Journal: The Journal of Cell Biology

Article Title: PLCγ1 promotes phase separation of T cell signaling components

doi: 10.1083/jcb.202009154

Figure Lengend Snippet: LAT cluster formation with Grb2, Sos1, and PLCγ1. Same condition as in Video 1 except that PLCγ1 (50 nM) was added together with Grb2 and Sos1 at 0 s. The video was recorded at a rate of 10 s/frame.

Article Snippet: Antibody , Anti-human PLCγ1 , Cell Signaling , Cat#5690, Western blot (1:4,000).

Techniques:

Journal: The Journal of Cell Biology

Article Title: PLCγ1 promotes phase separation of T cell signaling components

doi: 10.1083/jcb.202009154

Figure Lengend Snippet: Early phase of simulation of LAT cluster formation at low PLCγ1-to-LAT ratio. The simulation involves 200 LAT, 15 PLCγ1, 400 Grb2, and 200 Sos1 molecules, all in a monomeric state. The simulation starts at second 2.00, corresponding to timestep 0, and ends at timestep 10 × 10 6 . The interval between two frames is 0.2 × 10 6 timesteps, and the frame rate is 8 s −1 . Particle scheme is the same as in Fig. 6 A : gray, LAT; yellow, PLCγ1; blue, Grb2; and pink, Sos1.

Article Snippet: Antibody , Anti-human PLCγ1 , Cell Signaling , Cat#5690, Western blot (1:4,000).

Techniques:

Journal: The Journal of Cell Biology

Article Title: PLCγ1 promotes phase separation of T cell signaling components

doi: 10.1083/jcb.202009154

Figure Lengend Snippet: Full-length simulation of LAT cluster formation at low PLCγ1-to-LAT ratio. High-resolution video for visualizing individual chemical bonds. The simulation involves 200 LAT, 15 PLCγ1, 400 Grb2, and 200 Sos1 molecules, all in a monomeric state. The simulation starts at second 2.00, corresponding to timestep 0, and ends at timestep 50 × 10 6 . The interval between two frames is 0.2 × 10 6 timesteps, and the frame rate is 16 s −1 . Particle scheme is the same as in Fig. 6 A : gray, LAT; yellow, PLCγ1; blue, Grb2; and pink, Sos1.

Article Snippet: Antibody , Anti-human PLCγ1 , Cell Signaling , Cat#5690, Western blot (1:4,000).

Techniques:

Journal: The Journal of Cell Biology

Article Title: PLCγ1 promotes phase separation of T cell signaling components

doi: 10.1083/jcb.202009154

Figure Lengend Snippet: Early phase of simulation of LAT cluster formation at intermediate PLCγ1-to-LAT ratio. The simulation involves 200 LAT, 150 PLCγ1, 400 Grb2, and 200 Sos1 molecules, all in a monomeric state. The simulation starts at second 2.00, corresponding to timestep 0, and ends at timestep 10 × 10 6 . The interval between two frames is 0.2 × 10 6 timesteps, and the frame rate is 8 s −1 . Particle scheme is the same as in Fig. 6 A : gray, LAT; yellow, PLCγ1; blue, Grb2; and pink, Sos1.

Article Snippet: Antibody , Anti-human PLCγ1 , Cell Signaling , Cat#5690, Western blot (1:4,000).

Techniques:

Journal: The Journal of Cell Biology

Article Title: PLCγ1 promotes phase separation of T cell signaling components

doi: 10.1083/jcb.202009154

Figure Lengend Snippet: Full-length simulation of LAT cluster formation at intermediate PLCγ1-to-LAT ratio. High-resolution video for visualizing individual chemical bonds. The simulation involves 200 LAT, 150 PLCγ1, 400 Grb2, and 200 Sos1 molecules, all in a monomeric state. The simulation starts at second 2.00, corresponding to timestep 0, and ends at timestep 50 × 10 6 . The interval between two frames is 0.2 × 10 6 timesteps, and the frame rate is 16 s −1 . Particle scheme is the same as in Fig. 6 A : gray, LAT; yellow, PLCγ1; blue, Grb2; and pink, Sos1.

Article Snippet: Antibody , Anti-human PLCγ1 , Cell Signaling , Cat#5690, Western blot (1:4,000).

Techniques:

Journal: The Journal of Cell Biology

Article Title: PLCγ1 promotes phase separation of T cell signaling components

doi: 10.1083/jcb.202009154

Figure Lengend Snippet: Early phase of simulation of LAT cluster formation at high PLCγ1-to-LAT ratio. The simulation involves 200 LAT, 600 PLCγ1, 400 Grb2, and 200 Sos1 molecules, all in a monomeric state. The simulation starts at second 2.00, corresponding to timestep 0, and ends at timestep 10 × 10 6 . The interval between two frames is 0.2 × 10 6 timesteps, and the frame rate is 8 s −1 . Particle scheme is the same as in Fig. 6 A : gray, LAT; yellow, PLCγ1; blue, Grb2; and pink, Sos1.

Article Snippet: Antibody , Anti-human PLCγ1 , Cell Signaling , Cat#5690, Western blot (1:4,000).

Techniques:

Journal: The Journal of Cell Biology

Article Title: PLCγ1 promotes phase separation of T cell signaling components

doi: 10.1083/jcb.202009154

Figure Lengend Snippet: Full-length simulation of LAT cluster formation at high PLCγ1-to-LAT ratio. High-resolution video for visualizing individual chemical bonds. The simulation involves 200 LAT, 600 PLCγ1, 400 Grb2, and 200 Sos1 molecules, all in a monomeric state. The simulation starts at second 2.00, corresponding to timestep 0, and ends at timestep 50 × 10 6 . The interval between two frames is 0.2 × 10 6 timesteps, and the frame rate is 16 s −1 . Particle scheme is the same as in Fig. 6 A : gray, LAT; yellow, PLCγ1; blue, Grb2; and pink, Sos1.

Article Snippet: Antibody , Anti-human PLCγ1 , Cell Signaling , Cat#5690, Western blot (1:4,000).

Techniques:

Journal: The Journal of Cell Biology

Article Title: PLCγ1 promotes phase separation of T cell signaling components

doi: 10.1083/jcb.202009154

Figure Lengend Snippet: PLCγ1 promotes LAT cluster formation in vitro. (A) Domain structure of PLCγ1. (B) Schematics of the assay. (C) Top: TIRF microscopy revealed that both Grb2 and PLCγ1 (full-length) promote LAT microcluster formation. Alexa Fluor 488–labeled LAT at 300 molecules/µm 2 was incubated with 125 nM Sos1 and 250 nM Grb2 or PLCγ1 for 0.5 h before imaging. Scale bar, 5 µm. Bottom: Quantification of Grb2- or PLCγ1-driven LAT microclusters. LAT clustering was quantified as normalized variance . Shown are mean ± SD; n = 3 independent experiments. Unpaired two-tailed t test was used. *, P < 0.05; **, P < 0.01. (D) FRAP analysis revealed that PLCγ1-driven microclusters are less dynamic than Grb2-driven LAT microclusters. Shown are mean ± SD; n = 10 clusters.

Article Snippet: Antibody , Anti-human PLCγ1 , Cell Signaling , Cat#5690, Western blot (1:4,000).

Techniques: In Vitro, Microscopy, Labeling, Incubation, Imaging, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: PLCγ1 promotes phase separation of T cell signaling components

doi: 10.1083/jcb.202009154

Figure Lengend Snippet: Both the full-length and SH fragment of PLCγ1 drive LAT clustering. (A) TIRF microscopy revealed LAT microcluster formation with the full-length or SH2-SH2-SH3 domain of PLCγ1. Alexa Fluor 488–labeled, phosphorylated LAT at 300 molecules/µm 2 was incubated with 250 nM Sos1 and indicated concentrations of PLCγ1 or fragment. Scale bar, 5 µm. (B) Quantification of PLCγ1-driven LAT microclusters. Shown are mean ± SD; n = 3 independent experiments. (C) Recombinant proteins used in this study. Purified proteins were applied to SDS-PAGE, followed by Coomassie blue staining.

Article Snippet: Antibody , Anti-human PLCγ1 , Cell Signaling , Cat#5690, Western blot (1:4,000).

Techniques: Microscopy, Labeling, Incubation, Recombinant, Purification, SDS Page, Staining

Journal: The Journal of Cell Biology

Article Title: PLCγ1 promotes phase separation of T cell signaling components

doi: 10.1083/jcb.202009154

Figure Lengend Snippet: PLCγ1 cross-links LAT by two SH2 domains. (A) Domains of the proteins used in the study. (B) TIRF microscopy revealed that both nSH2 and cSH2 are required for PLCγ1-driven LAT microcluster formation. SH3 domain promotes cluster formation. Alexa Fluor 488–labeled LAT at 300 molecules/µm 2 was incubated with 300 nM Sos1 and 50 nM PLCγ1 for 0.5 h before imaging. Scale bar, 5 µm. (C) Quantification of PLCγ1-driven LAT microclusters. Shown are mean ± SD; n = 3 independent experiments. Unpaired two-tailed t test was used. *, P < 0.05. (D) Schematics of the assay of testing SH2 domain binding sites. (E) PLCγ1 nSH2 binds LAT Y132. Phosphopeptides were synthesized, biotinylated at the N terminus, and attached to the biotin-functionalized supported lipid bilayers by streptavidin. The SH2 domains were labeled with fluorescent dye (Maleimide-Ax647) and incubated with the individual phosphopeptides. The membrane-associated SH2 domain was measured by TIRF microscopy. Scale bar, 5 µm. (F) PLCγ1 cSH2 binds LAT Y171. Same settings as in E.

Article Snippet: Antibody , Anti-human PLCγ1 , Cell Signaling , Cat#5690, Western blot (1:4,000).

Techniques: Microscopy, Labeling, Incubation, Imaging, Two Tailed Test, Binding Assay, Synthesized, Membrane

Journal: The Journal of Cell Biology

Article Title: PLCγ1 promotes phase separation of T cell signaling components

doi: 10.1083/jcb.202009154

Figure Lengend Snippet: Domains required for PLCγ1-driven LAT clustering. (A) TIRF microscopy revealed LAT microcluster formation with a high concentration of PLCγ1 fragments. Alexa Fluor 488 LAT at 300 molecules/µm 2 was incubated with 125 nM Sos1 and 500 nM of indicated PLCγ1 fragments. Scale bar, 5 µm. (B) Quantification of LAT clustering in A. Shown are mean ± SD; n = 3 independent experiments. (C) TIRF microscopy revealed LAT microcluster formation in the presence or absence of PLCγ1. Alexa Fluor 488 LAT at 1,000 molecules/µm 2 was incubated with 500 nM Sos1 and 1,000 nM Grb2 with or without 100 nM full-length PLCγ1. Scale bar, 5 µm. (D) Quantification of LAT clustering. Shown are mean ± SD; n = 3 independent experiments. Unpaired two-tailed t test. ***, P < 0.001. (E) FRAP analysis revealed that PLCγ1 decreases the recovery of LAT signal in clusters after photobleaching. Shown are mean ± SD; n = 10 clusters.

Article Snippet: Antibody , Anti-human PLCγ1 , Cell Signaling , Cat#5690, Western blot (1:4,000).

Techniques: Microscopy, Concentration Assay, Incubation, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: PLCγ1 promotes phase separation of T cell signaling components

doi: 10.1083/jcb.202009154

Figure Lengend Snippet: Sos1 facilitates PLCγ1-driven LAT clustering. (A) TIRF microscopy revealed LAT microcluster formation with titrated PLCγ1. Alexa Fluor 488 LAT at 300 molecules/µm 2 was incubated with or without 250 nM Sos1 and the indicated concentration of PLCγ1 nSH2-cSH2-SH3 domains. Scale bar, 5 µm. (B) Quantification of LAT clustering in A. Shown are mean ± SD; n = 3 independent experiments. Unpaired two-tailed t test. *, P < 0.05; **, P < 0.01. (C) Binding affinity (Kd) of the SH3 domain of PLCγ1 to the PRM on Sos1 as measured by surface plasmon resonance. Shown are mean ± SEM. (D) TIRF microscopy revealed that SLP76 inhibits LAT microcluster formation driven by PLCγ1 and Sos1. Alexa Fluor 488 LAT at 300 molecules/µm 2 was incubated with 300 nM Sos1, 50 nM PLCγ1 (nSH2-cSH2-SH3 domains), and the indicated concentration of SLP76. Proteins were used at the physiologically relevant concentration. The cellular concentration of SLP76 was estimated as 150 nM (by MaxQB Database). Scale bar, 5 µm. (E) Quantification of LAT clustering in D. Shown are mean ± SD; n = 3 independent experiments. Unpaired two-tailed t test. *, P < 0.05; ns, not significant.

Article Snippet: Antibody , Anti-human PLCγ1 , Cell Signaling , Cat#5690, Western blot (1:4,000).

Techniques: Microscopy, Incubation, Concentration Assay, Two Tailed Test, Binding Assay, SPR Assay

Journal: The Journal of Cell Biology

Article Title: PLCγ1 promotes phase separation of T cell signaling components

doi: 10.1083/jcb.202009154

Figure Lengend Snippet: PLCγ1 cooperates with Grb2 to regulate LAT microcluster formation. (A) TIRF microscopy revealed that PLCγ1 regulates LAT microcluster formation in a nonmonotonic manner. Physiologically relevant concentrations of proteins were used in the assay: LAT at 300 molecules/µm 2 , Grb2 at 3 µM, Sos1 at 0.3 µM, and PLCγ1 at 50 nM. LAT was labeled with Alexa Fluor 488, PLCγ1 (SH2-2-3 domains) was labeled with DY547, and Sos1 was labeled with Alexa Fluor 647. Scale bar, 5 µm. (B) Quantification of LAT clustering, membrane recruitment of Sos1. Shown are mean ± SD; n = 3 independent experiments. Unpaired two-tailed t test was used. **, P < 0.01. (C) PLCγ1 accelerates LAT cluster formation. TIRF microscopy revealed the time course of LAT microcluster formation in the presence or absence of PLCγ1. LAT–Alexa Fluor 488 at 1,000 molecules/µm 2 was incubated with 1,000 nM Grb2 and 500 nM Sos1 and/or 50 nM PLCγ1 at time 0. Shown are mean ± SEM; n = 3 independent experiments.

Article Snippet: Antibody , Anti-human PLCγ1 , Cell Signaling , Cat#5690, Western blot (1:4,000).

Techniques: Microscopy, Labeling, Membrane, Two Tailed Test, Incubation

Journal: The Journal of Cell Biology

Article Title: PLCγ1 promotes phase separation of T cell signaling components

doi: 10.1083/jcb.202009154

Figure Lengend Snippet: A coarse-grained model explains how PLCγ1 nonmonotonically regulates LAT clustering. (A) Sketch of the model in which the proteins are represented as 2D particles decorated by interaction patches. All bonds possible in the system, based on biochemical data, are illustrated with colored lines. (B) Top: The average cluster size displays nonmonotonic dependence on the PLCγ1 concentration (gray circles). This behavior is well captured by the likelihood for cluster coalescence (black squares). Error bars represent statistical errors on the average size over 10 different realizations of the simulation, shown are mean ± SEM. Bottom: Snapshots of typical clusters in simulations, for relative PLCγ1:LAT concentrations of 0.075, 0.75, and 3 (these clusters contain, respectively, 19, 30, and 10 LAT molecules, and with reference to D, their compactness is 0.23, 0.39, and 0.48). (C) Breakdown of the coalescence likelihood per type of possible bond. The gray and pink areas represent available bonds involving a LAT or a Sos1 molecule, respectively; blue and yellow-orange bars represent bonds involving Grb2 and PLCγ1, respectively. (D) Compactness (gray circles, see Materials and methods) and fraction of terminal nodes (black squares), as a function of PLCγ1 concentration. Shown are mean ± SD; n = 10 realizations. (E) Fraction of LAT, PLCγ1, Sos1, and Grb2 molecules per cluster, as a function of PLCγ1 concentration, shaded according to the number of other molecules they are bound to.

Article Snippet: Antibody , Anti-human PLCγ1 , Cell Signaling , Cat#5690, Western blot (1:4,000).

Techniques: Concentration Assay

Journal: The Journal of Cell Biology

Article Title: PLCγ1 promotes phase separation of T cell signaling components

doi: 10.1083/jcb.202009154

Figure Lengend Snippet: Simulating PLCγ1’s effect on LAT cluster size and bond type. (A) The effect of PLCγ1 on LAT clustering is independent of LAT density ρ LAT . Coarse-grained model simulating LAT clustering as a function of PLCγ1 concentration. In a wide range of LAT densities tested, PLCγ1 regulates LAT clustering in a nonmonotonic manner. LAT clusters are quantified by the number of LAT in each cluster (solid line) or the total number of molecules (LAT, Grb2, PLCγ1, or Sos1) in each cluster (dashed line, scaled down by a factor of 4 to fit in the same plot). Surface densities ρ LAT are in units of σ −2 , where σ is the diameter of a particle, of the order of a few nanometers, and our experiments correspond roughly to ρLAT = 0.02σ −2 (see Materials and methods and Simulation details). Shown are mean ± SEM. (B) Average coordination number for all four kinds of particles, as a function of ratio of PLCγ1:LAT, broken down to the contribution of each specific bond. Yellow-orange bars represent bonds involving PLCγ1, and blue bars, Grb2; a gray background represents bonds involving LAT, and a pink background, Sos1. Here, as throughout , ρ LAT = 0.02 σ −2 . See , section SI 3, for a complete analysis. Shown are mean ± SEM.

Article Snippet: Antibody , Anti-human PLCγ1 , Cell Signaling , Cat#5690, Western blot (1:4,000).

Techniques: Concentration Assay

Journal: The Journal of Cell Biology

Article Title: PLCγ1 promotes phase separation of T cell signaling components

doi: 10.1083/jcb.202009154

Figure Lengend Snippet: PLCγ1 promotes LAT clustering, SLP76 phosphorylation, and ERK activation in Jurkat T cells. (A) Diminished LAT microcluster formation in PLCγ1-null cells. Wild-type or PLCγ1-null Jurkat T cells expressing LAT-mCherry were plated on OKT3-coated cover glass. LAT microcluster formation was revealed by TIRF microscopy. Images showed clustering 90 s after cell landing on the glass. Scale bar, 5 µm. Shown are mean ± SEM; n = 25 or 26 cells. Unpaired two-tailed t test was used. **, P < 0.01. (B) The nSH2 and SH3 domain of PLCγ1 promotes LAT cluster formation. PLCγ1-null Jurkat T cells expressing LAT-mCherry were reconstituted with the GFP-tagged wild-type, Δ nSH2, or Δ SH3 PLCγ1. Those cells were plated on OKT3-coated cover glass. Images showed clustering 90 s after cell landing on the glass. LAT microcluster formation was revealed by TIRF microscopy. Scale bar, 5 µm. Shown are mean ± SEM; n = 22–30 cells. Unpaired two-tailed t test was used. **, P < 0.01. (C) Immunoblot analysis of LAT-null Jurkat T cells reconstituted with the GFP-tagged wild-type, Δ nSH2, or Δ SH3 PLCγ1. Cells were stimulated with 2 µg/ml anti-CD3 and anti-CD28 antibodies for 2 min, lysed, and applied for Western blot analysis. MW, molecular weight. (D) Quantification of the level of indicated proteins, after being normalized to the expression level of GAPDH. Shown are mean ± SD; n = 3 independent experiments. Unpaired two-tailed t test was used. *, P < 0.05; **, P < 0.01.

Article Snippet: Antibody , Anti-human PLCγ1 , Cell Signaling , Cat#5690, Western blot (1:4,000).

Techniques: Phospho-proteomics, Activation Assay, Expressing, Microscopy, Two Tailed Test, Western Blot, Molecular Weight

Journal: The Journal of Cell Biology

Article Title: PLCγ1 promotes phase separation of T cell signaling components

doi: 10.1083/jcb.202009154

Figure Lengend Snippet: The nSH2–pY interaction is required for PLCγ1-mediated LAT clustering and signaling. (A) Diminished LAT microcluster formation in cells expressing PLCγ1 R586K. R586K abolishes the nSH2 interaction with LAT pY132. PLCγ1-null Jurkat T cells that express LAT-mCherry were reconstituted with the GFP-tagged wild type or R586K PLCγ1. They were plated on OKT3-coated cover glass. LAT microcluster formation was revealed by TIRF microscopy. Left: Images showed clustering 60 s after cell landing on the glass. Scale bar, 5 µm. Right: Quantification of clustering. Shown are mean ± SEM; n = 23–29 cells. Unpaired two-tailed t test was used. *, P < 0.05. (B) Immunoblot analysis of LAT-null Jurkat T cells reconstituted with the GFP-tagged wild type or R586K PLCγ1. Cells were stimulated with 2 µg/ml anti-CD3 and anti-CD28 antibodies for 2 min, lysed, and applied for Western blot analysis. MW, molecular weight. (C) Quantification of the level of indicated proteins, after being normalized to the expression level of GAPDH. Shown are mean ± SD; n = 3 independent experiments. Unpaired two-tailed t test was used. *, P < 0.05; ***, P < 0.001.

Article Snippet: Antibody , Anti-human PLCγ1 , Cell Signaling , Cat#5690, Western blot (1:4,000).

Techniques: Expressing, Microscopy, Two Tailed Test, Western Blot, Molecular Weight

Journal: The Journal of Cell Biology

Article Title: PLCγ1 promotes phase separation of T cell signaling components

doi: 10.1083/jcb.202009154

Figure Lengend Snippet: The lipase-independent signaling role of PLCγ1. (A) Immunoblot analysis of LAT-null Jurkat T cells reconstituted with the GFP-tagged wild type or H380F PLCγ1. H380F abolishes most of the enzymatic activity of PLCγ1. Cells were stimulated with 2 µg/ml anti-CD3 and anti-CD28 antibodies for 2 min, lysed, and applied for Western blot analysis. MW, molecular weight. (B) Quantification of the level of indicated proteins, after being normalized to the expression level of GAPDH. Shown are mean ± SD; n = 3 independent experiments. Unpaired two-tailed t test was used. **, P < 0.01. (C) Cells as indicated were stimulated with 2 µg/ml anti-CD3 and anti-CD28 antibodies for 2 min, lysed, and applied for Western blot analysis. (D) The level of LAT pY171, after being normalized to the level of GAPDH, was quantified. Shown are mean ± SD; n = 3 independent experiments.

Article Snippet: Antibody , Anti-human PLCγ1 , Cell Signaling , Cat#5690, Western blot (1:4,000).

Techniques: Western Blot, Activity Assay, Molecular Weight, Expressing, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: PLCγ1 promotes phase separation of T cell signaling components

doi: 10.1083/jcb.202009154

Figure Lengend Snippet: PLCγ1 protects LAT from dephosphorylation by CD45. (A) Reduced phosphorylation at LAT Y132 in PLCγ1-null cells. Cells as indicated were stimulated with 2 µg/ml anti-CD3 and anti-CD28 antibodies for 2 min, lysed, and applied for Western blot analysis. The level of indicated proteins, after being normalized to the level of GAPDH, was quantified. Shown are mean ± SD; n = 3 independent experiments. Unpaired two-tailed t test was used. **, P < 0.01. MW, molecular weight. (B) PLCγ1 prevents LAT Y132 from being dephosphorylated. Cells as indicated were pretreated with 0.1 mM vanadate (pan-phosphatase inhibitor) before being stimulated with 2 µg/ml anti-CD3 and anti-CD28 antibodies for 2 min, lysed, and applied for Western blot analysis. The level of indicated proteins, after being normalized to the level of GAPDH, was quantified. Shown are mean ± SD; n = 3 independent experiments. Unpaired two-tailed t test was used. **, P < 0.01; ns, not significant. (C) Schematics of the in intro dephosphorylation assay. (D) PLCγ1 prevents LAT Y132 from being dephosphorylated by CD45 in vitro. pLAT, at 1,000 molecules/µm 2 , was incubated with 1 µM Grb2, 0.5 µM Sos1, and/or 100 nM full-length PLCγ1 for 0.5 h. CD45 was then added to dephosphorylate pLAT for 5 min. The reaction was terminated by adding SDS-PAGE loading buffer with 2 mM vanadate. The level of phosphorylated LAT, after being normalized to total LAT, was quantified. Shown are mean ± SD; n = 3 independent experiments.

Article Snippet: Antibody , Anti-human PLCγ1 , Cell Signaling , Cat#5690, Western blot (1:4,000).

Techniques: De-Phosphorylation Assay, Phospho-proteomics, Western Blot, Two Tailed Test, Molecular Weight, In Vitro, Incubation, SDS Page

Journal: The Journal of Cell Biology

Article Title: PLCγ1 promotes phase separation of T cell signaling components

doi: 10.1083/jcb.202009154

Figure Lengend Snippet: Mechanism of phosphotyrosine protection by LAT clustering. (A) TIRF microscopy revealed clustered and unclustered LAT and Grb2. Alexa Fluor 488–labeled LAT at 1,000 molecules/µm 2 was incubated with 1 µM Grb2 + 500 nM Sos1 or 6 µM Grb2. 20% of Grb2 was labeled with Alexa Fluor 568. Similar Grb2 was recruited to the membrane in the two indicated conditions. Shown are mean ± SD; n = 3 independent experiments. Scale bar, 5 µm. (B) LAT clustering by Grb2 prevents LAT Y132 from being dephosphorylated by CD45 in vitro. pLAT, at 1,000 molecules/µm 2 , was incubated with 1 µM Grb2, 0.5 µM Sos1, or 1 µM Grb2. CD45 was added to dephosphorylate pLAT for 5 min. The reaction was terminated by adding SDS-PAGE loading buffer with 2 mM vanadate. The level of LAT pY132, after being normalized to total LAT, was quantified. Shown are mean ± SD; n = 3 independent experiments. MW, molecular weight. (C) CD45 is excluded by Grb2- or PLCγ1-mediated LAT clustering. pLAT–Alexa Fluor 488 (1,000 molecules/μm 2 ) was incubated with 1 µM Sos1 and 1 µM Grb2 or 1 µM PLCγ1-SH2-2-3 fragment. The cytoplasmic domain of CD45-TMR (4 nM, with an N-terminal His10 tag) was added, and its localization was visualized by TIRF microscopy. Scale bar, 5 µm. (D) Quantification of fluorescence intensity of pLAT and CD45 along the line scan indicated by a white line in the top merged image.

Article Snippet: Antibody , Anti-human PLCγ1 , Cell Signaling , Cat#5690, Western blot (1:4,000).

Techniques: Microscopy, Labeling, Incubation, Membrane, In Vitro, SDS Page, Molecular Weight, Fluorescence

Journal: The Journal of Cell Biology

Article Title: PLCγ1 promotes phase separation of T cell signaling components

doi: 10.1083/jcb.202009154

Figure Lengend Snippet: Key resources

Article Snippet: Antibody , Anti-human PLCγ1 , Cell Signaling , Cat#5690, Western blot (1:4,000).

Techniques: Cell Culture, Expressing, Knock-Out, CRISPR, Plasmid Preparation, Infection, Bacteria, Recombinant, Western Blot