plcγ1 y783  (Cell Signaling Technology Inc)

Journal: Cellular signalling

Article Title: Phosphoproteomic analysis of successive Jurkat CD19-CAR generations reveals TCRζ-driven signalling

doi: 10.1016/j.cellsig.2025.112204

Figure Lengend Snippet: Expression of CD19-CARs and phosphoproteomic workflow. (A) Schematic representation of the cell lines used in this study. (B) Flow cytometry analysis showing total GFP expression in CD19-CAR Jurkats. (C-D) Western blot analysis of co-culture samples showing phosphorylation of Erk1/2 and PLCγ1, respectively. (E) Flow chart depicting the co-culture stimulation phosphoproteomics workflow used in this study. Details are in the Methods section.

Article Snippet: Primary Antibodies: 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), Lck (1:4000, Cell Signaling Technologies #2657), Src family kinase activation site (Src Y416 ; 1:2000, Cell Signaling Technologies #2101).

Techniques: Expressing, Flow Cytometry, Western Blot, Co-Culture Assay, Phospho-proteomics

Journal: Cellular signalling

Article Title: Phosphoproteomic analysis of successive Jurkat CD19-CAR generations reveals TCRζ-driven signalling

doi: 10.1016/j.cellsig.2025.112204

Figure Lengend Snippet: PTP activity globally, but not TCR-specific regulatory PTPs, restrict CD69 expression in a costimulation-dependent manner. (A) Simplified schematic representation of CAR T cell signalling with inhibitors and their targets noted. (B-D) Western blot analysis of PLCγ1 Y783 , Lck Y394 , and Erk1 T202Y204 /Erk2 T185Y187 , respectively, in samples treated with PTPN22-IN-1, TPI-1, or PV. One sample is shown here for display. Three replicates and quantification are shown in Supporting Figure 7 . (E-F) Dotplot showing the effect of PTPN22-IN-1, TPI-1, and PV on the percentage of CD69+ WT Jurkats in non co-culture and co-culture samples, respectively. (G-H) As in E and F, except for ζ-CAR Jurkats. (I-J) As in E and F, except for 28ζ-CAR Jurkats. (K-L) As in E and F, except for BBζ-CAR Jurkats. (M-N) As in E and F, except for 28BBζ-CAR Jurkats. Three biological replicates are shown for each flow cytometry experiment.

Article Snippet: Primary Antibodies: 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), Lck (1:4000, Cell Signaling Technologies #2657), Src family kinase activation site (Src Y416 ; 1:2000, Cell Signaling Technologies #2101).

Techniques: Activity Assay, Expressing, Western Blot, Co-Culture Assay, Flow Cytometry

Journal: Cellular signalling

Article Title: Phosphoproteomic analysis of successive Jurkat CD19-CAR generations reveals TCRζ-driven signalling

doi: 10.1016/j.cellsig.2025.112204

Figure Lengend Snippet: Pharmacological inhibition of Itk with Soquelitinib reduces baseline CD69 expression without impairing CAR T cell activation. (A) Simplified schematic representation of CAR T cell signalling with inhibitors and their targets noted. (B-D) Western blot analysis of PLCγ1 Y783 , Lck Y394 , and Erk1 T202Y204 /Erk2 T185Y187 , respectively, in samples treated with PP1, Soquelitinib, or U0126. One sample is shown here for display. Three replicates and quantification are shown in Supporting Figure 8 . (E-F) Dotplot showing the effect of PP1, Soquelitinib, and U0126 on the percentage of CD69+ WT Jurkats in non co-culture and co-culture samples, respectively. (G-H) As in E and F, except for ζ-CAR Jurkats. (I-J) As in E and F, except for 28ζ-CAR Jurkats. (K-L) As in E and F, except for BBζ-CAR Jurkats. (M-N) As in E and F, except for 28BBζ-CAR Jurkats. Three biological replicates are shown for each flow cytometry experiment.

Article Snippet: Primary Antibodies: 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), Lck (1:4000, Cell Signaling Technologies #2657), Src family kinase activation site (Src Y416 ; 1:2000, Cell Signaling Technologies #2101).

Techniques: Inhibition, Expressing, Activation Assay, Western Blot, Co-Culture Assay, Flow Cytometry

Journal: Scientific Reports

Article Title: The phosphatases TCPTP, PTPN22, and SHP1 play unique roles in T cell phosphotyrosine maintenance and feedback regulation of the TCR

doi: 10.1038/s41598-025-12951-2

Figure Lengend Snippet: Knockout of PTPN2 (TCPTP), PTPN22 (PTPN22), or PTPN6 (SHP1) retain TCR signalling and responsiveness to U0126 treatment. ( A ) Truncated schematic of TCR signalling, with black lines/arrows showing forward signalling and red lines/arrows showing the effect of U0126 treatment to reduce flux through the TCR signalling pathway. ( B ) Representative Western blots targeting Erk1 T202Y204 /Erk2 T185Y187 during a U0126 titration. Quantification of U0126 titration Western blots targeting ( C ) Erk1 T202Y204 /Erk2 T185Y187 and ( D ) pY. ( E ) Representative Western blots targeting Erk1 T202Y204 /Erk2 T185Y187 during a TCR stimulation time course in the presence or absence of U0126. Quantification of time course Western blots targeting ( F ) Erk1 T202Y204 /Erk2 T185Y187 , ( G ) pY, ( H ) Src family kinase activation site, sequence NEpYTAR, and ( I ) PLCγ1 Y783 . For all Western blots, statistical significance between 0-min normalised, log2 transformed abundances is determined by Fisher’s LSD with the Holm Sidak correction. ♠♣♥♦ indicate p < 0.05 when comparing the given time point to the 0-min sample for Jurkat, J.TCPTP-, J.PTPN22-, and J.SHP1-, respectively. Upper symbols are for 0.04% DMSO treatment, and lower symbols are for U0126 treatment time courses. `n.s’ indicates no statistical significance in that comparison for any cell line.

Article Snippet: Western Primary Antibodies included pY clone 4G10 (1:2000, Cell Signalling Technologies #96215), PLCγ1 (1:10,000, Millipore Sigma #05-163), phosphorylated PLCγ1 Y783 (1:2000, Cell Signalling Technologies #2821), Erk1/2 (Cell Signalling Technologies #9107), phosphorylated Erk T202Y204 (1:2000, Cell Signalling Technologies #9101), Lck (1:5000, Cell Signalling Technologies #2657S), phosphorylated Src Family Kinase Y416 (1:2000, Cell Signalling Technologies #59548S, 2101S), TCPTP (1:2000, Cell Signalling Technologies #58935S), PTPN22 (1:1000, Thermo #11783–1-AP), SHP1 (1:500, Santa Cruz Biotechnology #sc-287), and Vav1 (1:2000, Cell Signalling Technologies #4657S, 2505S).

Techniques: Knock-Out, Western Blot, Titration, Activation Assay, Sequencing, Transformation Assay, Comparison