plcγ1 y783  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: Evaluating First-Pass, High Protein Capacity Desalting Techniques For Phosphoproteomics Applications

doi: 10.1101/2025.06.03.657744

Figure Lengend Snippet: Samples prepared using ProtiFi S-Trap show superior identification of biologically relevant, T cell receptor responsive pY sites. (A) Simplified schematic of T cell receptor signalling. (B-E) Volcano plot analysis comparing pY site abundance between 2 minutes and 0 minutes of stimulation for samples prepared using Waters Sep-Pak C18 columns, Pierce C18 spin columns, TECAN WWP2 NBE columns, and ProtiFi S-Trap spin columns, respectively. Values to the left indicate the number of unique pY sites in a given threshold (0.1 > q ≥ 0.05, 0.05 > q ≥ 0.01, 0.01 > q from bottom to top). (F) Western blot analysis of PLCγ1 Y783 with accompanying quantification. *** indicates p < 0.001 by a Welch’s T-test. (G) Heatmaps comparing 2 minutes and 0 minutes of T cell receptor stimulation for unique pY sites. Grey squares indicate n ≤ 3 replicates for a given pY site in either 2- or 0-minute groups. * indicates 0.1 > q ≥ 0.05, ** indicates 0.05 > q ≥ 0.01, *** indicates 0.01 > q.

Article Snippet: Primary antibodies: pY clone 4G10 (1:2000, Cell Signalling Technologies #96215), GAPDH (1:10000, Millipore Sigma #G9545), PLCγ1 (1:10000, Millipore Sigma #05-163), PLCγ1 Y783 (1:2000, Cell Signalling Technologies #2821), Erk1/2 (Cell Signalling Technologies #9107), Erk T202Y204 (1:2000, Cell Signalling Technologies #9101) Secondary antibodies: IRDye 680RD Goat anti-Mouse IgG (LI-COR #926-68070) IRDye 800CW Goat anti-Mouse IgG (LI-COR #926-33210), IRDye 680RD Donkey anti Rabbit IgG (LI-COR #926-68073), IRDye 800CW Donkey anti Rabbit IgG (LI-COR #926-32213)

Techniques: Western Blot

Journal: Journal for immunotherapy of cancer

Article Title: Cbl-b inhibitor NX-1607 activates MAPK/ERK signaling pathway and enhances T-cell activation.

doi: 10.1136/jitc-2024-011180

Figure Lengend Snippet: Figure 1 Impact of NX-1607 on Cbl-b activity and T-cell activation/proliferation. (a) The chemical structure of the Cbl-b inhibitor NX-1607. (b) Illustration of the HTRF assay showing Cbl-b activity inhibition by a Cbl-b inhibitor, involving SRC, UBE1 (E1), UbcH5b (E2), Cbl-b (E3), ubiquitin, and ATP. Image was created with BioRender.com, with permission. (c) IC50 values of NX-1607 for inhibiting Cbl-b activity were determined using HTRF assays, with data from two biological replicates presented as mean±SEM. (d) NX-1607 increases p-PLCγ1-Y783 and p-HCLS1-Y397 in Jurkat T cells in a concentration-dependent manner. Cells were treated with NX-1607 or plate-bound anti-CD3 for 6 hours, followed by western blot analysis. (e) Histograms showing CD69 expression in Jurkat T and human primary CD3+ T cells after NX-1607 treatment, with or without CD3 stimulation. Cells were left untreated or pretreated with 2 μM NX-1607 for 1 hour, then exposed to plate-bound anti-CD3 for an additional 24 hours and analyzed by flow cytometry. (f) Mean fluorescence intensity quantification of CD69 in T cells in (e), based on three independent experiments, presented as mean±SD, with one-way analysis of variance analysis (***p<0.001). (g) IL2 and IFNG messenger RNA levels in primary human CD3+ T cells after 2 μM NX-1607 treatment, with or without anti-CD3 stimulation (2 µg/ mL) for 48 hours. Data from three independent experiments, presented as mean±SD, with t-test analysis (***p<0.001, **p<0.01). (h) The proliferative effects of NX-1607 were assessed in primary human CD3+, CD4+, and CD8+ T cells, with or without anti-CD3 stimulation (2 µg/mL) for 72 hours. The upper panel presents OD450 readings from primary human CD3+, CD4+, and CD8+ T- cell populations, while the lower panel illustrates the corresponding cell counts for each T-cell subset. Data obtained from three independent experiments (mean±SD, t-test) (*p<0.05, **p<0.01, ***p<0.001). Cbl-b, casitas B lymphoma-b; HTRF, homogeneous time-resolved fluorescence; IC50, half-maximal inhibitory concentration.

Article Snippet: Detection of phosphoPLCγ1 was performed by incubating the samples with a rabbit antibody specific to phospho- PLCγ1 (#14008, CST) for 2 hours at room temperature, followed by a 1- hour incubation with Goat anti- Rabbit Alexa Fluor 488 (#A11008, Invitrogen) at room temperature.

Techniques: Activity Assay, Activation Assay, HTRF Assay, Inhibition, Ubiquitin Proteomics, Concentration Assay, Western Blot, Expressing, Flow Cytometry, Fluorescence

Journal: Journal for immunotherapy of cancer

Article Title: Cbl-b inhibitor NX-1607 activates MAPK/ERK signaling pathway and enhances T-cell activation.

doi: 10.1136/jitc-2024-011180

Figure Lengend Snippet: Figure 4 MAPK/ERK signaling pathway is crucial for NX-1607-induced T-cell activation. (a) Concentration-dependent impact of NX-1607 on p-MEK1/2, p-ERK1/2, p-PLCγ1, and p-HCLS1 in both Jurkat T and HuT78 cells. (b) Western blot analysis of p-MEK1/2, p-ERK1/2, p-PLCγ1, and p-HCLS1 in Jurkat T cells treated with different Cbl-b inhibitors, including NX-1607 and Cbl-b-IN1, for 6 hours. (c) Combining NX-1607 treatment with CD3 stimulation for 6 hours increased p-MEK, p-ERK, p-PLCγ1, and p-HCLS1 levels, in both primary mouse and human CD3+ T cells. (d) The messenger RNA levels of IL2 and IFNG in primary human CD3+ T cells after 48 hours of 2 μM NX-1607 treatment, with or without 2 µM MEK/ERK inhibitors and 2 µg/mL anti-CD3 stimulation. Data were obtained from two biological replicates. (e) Ki-67 expression in primary mouse CD3+ T cells after 72 hours of NX-1607 treatment, with or without MEK/ERK inhibitors and CD3 stimulation, as analyzed by flow cytometry. In the right panel, MEKi-trametinib (blue) and GDC0623 (purple); ERKi-ulixertinib (magenta) and LY3214996 (orange). Data from three independent experiments; statistical analysis was performed by one-way analysis of variance. ***p<0.001. Cbl-b, casitas B lymphoma-b.

Article Snippet: Detection of phosphoPLCγ1 was performed by incubating the samples with a rabbit antibody specific to phospho- PLCγ1 (#14008, CST) for 2 hours at room temperature, followed by a 1- hour incubation with Goat anti- Rabbit Alexa Fluor 488 (#A11008, Invitrogen) at room temperature.

Techniques: Activation Assay, Concentration Assay, Western Blot, Expressing, Flow Cytometry

Journal: Journal for immunotherapy of cancer

Article Title: Cbl-b inhibitor NX-1607 activates MAPK/ERK signaling pathway and enhances T-cell activation.

doi: 10.1136/jitc-2024-011180

Figure Lengend Snippet: Figure 5 Effects of the SRC-PLCγ1-MEK/ERK pathway on NX-1607-induced T-cell activation. (a and b) Western blot analysis of p-MEK1/2, p-ERK1/2, p-PLCγ1, and p-HCLS1 levels after NX-1607 treatment combined with MEK/ERK inhibitors (#1: trametinib, #2: GDC-0623, #3: ulixertinib, #4: LY3214996) or MAPK3/1 (ERK1/2) double KO via CRISPR in Jurkat T cells. (c) Flow cytometry histograms and MFI quantification of CD69 expression in parental and MAPK3/1 (ERK1/2) KO Jurkat T cells after 24-hour NX-1607 exposure. (d) Western blot of p-MEK1/2, p-ERK1/2, p-PLCγ1, and p-HCLS1 in Jurkat T cells treated with NX-1607 for 6 hours, with or without SRC inhibitors (dasatinib#1; saracatinib#2). (e) IL2 and IFNG mRNA levels in Jurkat T cells treated with NX-1607, with or without CD3 stimulation, and with or without SRC inhibitors (dasatinib#1; saracatinib#2) treatment for 48 hours. All data obtained from three biological replicates, presented as mean±SD, with statistical analysis by one-way ANOVA. ***p<0.001. (f) The concentration-dependent effects of the PLC inhibitor (U-73122) on p-MEK1/2, p-ERK1/2, and p- PLCγ1 levels in Jurkat T cells treated with NX-1607 for 6 hours, as determined by western blotting. (g) Deletion of PLCG1 in Jurkat T cells led to reduced p-MEK1/2 and p-ERK1/2 levels after 6 hours of NX-1607 treatment. (h) Flow cytometry histograms and MFI quantification of CD69 expression in parental and PLCG1 KO Jurkat T cells after 24-hour NX-1607 exposure. All data are from three biological replicates, presented as mean±SD, with statistical analysis by one-way ANOVA. ***p<0.001. ANOVA, analysis of variance; MFI, mean fluorescence intensity; mRNA, messenger RNA.

Article Snippet: Detection of phosphoPLCγ1 was performed by incubating the samples with a rabbit antibody specific to phospho- PLCγ1 (#14008, CST) for 2 hours at room temperature, followed by a 1- hour incubation with Goat anti- Rabbit Alexa Fluor 488 (#A11008, Invitrogen) at room temperature.

Techniques: Activation Assay, Western Blot, CRISPR, Flow Cytometry, Expressing, Concentration Assay, Fluorescence

Journal: Journal for immunotherapy of cancer

Article Title: Cbl-b inhibitor NX-1607 activates MAPK/ERK signaling pathway and enhances T-cell activation.

doi: 10.1136/jitc-2024-011180

Figure Lengend Snippet: Figure 6 NX-1607 significantly inhibits A20 xenograft tumor growth and boosts T-cell infiltration. (a) NX-1607’s impact on RTV and body weight in the A20 model. Tumor-bearing BALB/c mice received 60 mg/kg NX-1607 or vehicle orally once daily for 14 days. Data are mean±SEM, analyzed by two-way analysis of variance. ***p<0.001. (b) Representative images and mean±SD tumor weights after 14 days of vehicle or 60 mg/kg NX-1607 treatment. (c) Phosphorylated PLCγ1 and ERK1/2 levels were assessed in circulating T cells from mice (n=3 per group) treated with a single oral dose of NX-1607 (60 mg/kg) or vehicle. Whole blood was collected and incubated with 2 µg/mL anti-CD3 and 2 µg/mL anti-CD28 for 1 hour, and p-PLCγ1 and p-ERK1/2 levels were measured in purified CD3+ T cells using flow cytometry. Statistical significance was assessed using t-test (**p<0.01). (d) Following 14 days of treatment with either vehicle or NX-1607 (60 mg/kg), tumors were harvested and analyzed for CD45, CD3, CD4, CD8, CD69, and Ki-67 expression via flow cytometry. Data were shown as mean±SD and statistical analysis was assessed using t-test (*p<0.05). (e) A20 tumors were collected after 14 days of treatment with either vehicle or NX-1607 (60 mg/kg), then fixed, and analyzed via IHC for CD3, CD4, and CD8 expression. Data are presented as mean±SD, with significance determined by t-test (***p<0.001). IHC, Immunohistochemistry; MFI, mean fluorescence intensity.

Article Snippet: Detection of phosphoPLCγ1 was performed by incubating the samples with a rabbit antibody specific to phospho- PLCγ1 (#14008, CST) for 2 hours at room temperature, followed by a 1- hour incubation with Goat anti- Rabbit Alexa Fluor 488 (#A11008, Invitrogen) at room temperature.

Techniques: Incubation, Purification, Flow Cytometry, Expressing, Immunohistochemistry, Fluorescence

Journal: Journal for immunotherapy of cancer

Article Title: Cbl-b inhibitor NX-1607 activates MAPK/ERK signaling pathway and enhances T-cell activation.

doi: 10.1136/jitc-2024-011180

Figure Lengend Snippet: Figure 7 Graphic model depicting the mechanism of NX-1607 in enhancing T-cell activation. The schematic demonstrates that NX-1607 inhibits Cbl-b, thereby lowering the activation threshold of TCR signaling. This results in increased T-cell activation via enhanced phosphorylation of PLCγ1 and subsequent activation of the MAPK/ERK pathway. The outcome is a robust T-cell response, characterized by increased proliferation, elevated expression of CD69, and upregulation of IL2 and IFNG mRNA levels, alongside significantly enhanced antitumor activity. The image was created using BioRender.com, with permission. Cbl-b, casitas B lymphoma-b; mRNA, messenger RNA; TCR, T-cell receptor.

Article Snippet: Detection of phosphoPLCγ1 was performed by incubating the samples with a rabbit antibody specific to phospho- PLCγ1 (#14008, CST) for 2 hours at room temperature, followed by a 1- hour incubation with Goat anti- Rabbit Alexa Fluor 488 (#A11008, Invitrogen) at room temperature.

Techniques: Activation Assay, Phospho-proteomics, Expressing, Activity Assay