a770041  (MedChemExpress)

Journal: The Journal of Biological Chemistry

Article Title: Role of the membrane anchor in the regulation of Lck activity

doi: 10.1016/j.jbc.2022.102663

Figure Lengend Snippet: Dynamic maintenance of the Lck A pool . A , schematics of the generation and maintenance of Lck isoforms at the PM. From left to right : inactive (Lck I ), primed (Lck P ), active (Lck A ), active-double phosphorylated (Lck ADP ). CD45 is in large stoichiometric excess (>>) over Lck. B , Left , 3D-SIM of Lck ( green ) in CD4 + T cells or JCaM1.6 cells expressing Lck or LckΔSH4. Scale bars ( white ). PM and nucleus are neatly defined by CD45 ( red ) and DAPI staining ( blue ), respectively. Right , histograms of the ratio of Lck or LckΔSH4 amounts detected at PM and in CP (PM/CP). Error bars: SD for n ≥ 10 cells of three or more independent experiments. Unpaired t test: p > 0.5 (non-significant, ns) for CD4 + T cells versus JCaM1.6-Lck; ∗∗∗∗ p < 0.0001 for CD4 + T cells versus LckΔSH4. C , Left , 3D-SIM of pY394-Lck ( green ) in CD4 + T cells or in JCaM1.6 expressing Lck. Right , histograms of PM/CP ratio of pY394 in CD4 + T cells or in JCaM1.6 expressing Lck. Error bars: SD for n ≥ 10 cells from three or more independent experiments. Unpaired t test, ∗∗∗∗ p < 0.0001. D , Left , representative FCM of Lck A in Cln20 cells treated ( red ) with 2 μM A770041 or carrier (DMSO, blue ) at 37 °C for 30 s or 5 min. JCaM1.6 ( gray ), negative control to set pY416 antibody (Ab) background. Right, histogram of mean ± SD of Lck A (% of inhibition), n = 3. Unpaired t test, ∗∗∗∗ p < 0.0001. E , Left , representative FCM of Lck A in Clone 20 cells reacted ( green ) or not ( blue ) with 100 μM catalase-treated pervanadate (PV) at 37 °C for 1 min. JCaM1.6 ( gray ), negative control for pY416 Ab background. Right , histogram of mean ± SEM of Lck A n = 2, unpaired t test, ∗∗ p < 0.01. F , Left , representative FCM of pY505-Lck in Jurkat cells treated ( red ) with 5 μM A770041 or carrier (DMSO, blue ) at 37 °C for 5 min. JCaM1.6 ( gray ) negative control for pY505-Lck Ab background. Right , histogram of mean ± SD of Lck A (% of inhibition), n = 4, unpaired t test, ∗∗∗∗ p < 0.0001. G , Left , 3D-SIM of pY505-Lck ( green ) in CD4 + T cells or in JCaM1.6 expressing Lck or LckΔSH4. Right , histogram of PM/CP ratio for pY505 in CD4 + T cells or in JCaM1.6 expressing Lck or LckΔSH4. Error bars: SD for n ≥ 10 cells from three or more independent experiments, p > 0.5 (non-significant, ns). 3D-SIM, 3D structured illumination microscopy; CP, cytoplasmic; FCM, flow cytometry; Lck A , active form of Lck; PM, plasma membrane; LckΔSH4, Lck-lacking SH4.

Article Snippet: A770041 (Axon Medichem), Sodium Orthovanadate (Vanadate) New England BioLabs (NEB), catalase, and hydrogen peroxide (30%) are from Sigma-Aldrich.

Techniques: Expressing, Staining, Negative Control, Inhibition, Microscopy, Flow Cytometry

Journal: Nature Immunology

Article Title: Unconventional human CD61 pairing with CD103 promotes TCR signaling and antigen-specific T cell cytotoxicity

doi: 10.1038/s41590-024-01802-3

Figure Lengend Snippet: a , Representative dot plot showing PCC of TCRαβ and CD61 colocalization (left) and TCRαβ and CD103 colocalization (right) at 5, 10 and 15 min after synaptic formation. P value (TCRαβ-CD61, 5 versus 15 min): 0.0009, P value (TCRαβ-CD103, 5 versus 10 min): 0.0075, P value (TCRαβ-CD103, 5 versus 15 min): 0.000782. Each dot represents the average PCC per synapse. n = 150 cells examined over three independent experiments; each dot represents the average PCC per synapse. Time of 5 min: 91 synapses; time of 10 min: 101 synapses; time of 15 min: 71 synapses. b , c , Average MFI of either Zap70 (pY292) or PLCγ1 (pY783) on ( b ) WT CD61 + T cell clone (from patient 1) following treatment with either 25, 50 or 100 nM CD61 siRNA, or no treatment ( b ), as well as on WT CD61 + , CD61 KO or WT CD61 + T cell clones (from patient 1), by flow cytometry ( c ). d , Bar graph showing the average MFI of Zap70 (pY292) on CD61 + T cell lines, following treatment with αCD61 (neutralizing treatment), IgG isotype control treatment or no treatment, by flow cytometry. P values: (patient 1: 0.00076, patient 2: 0.0083, patient 3: 0.0096, patient 4: 0.00031, patient 5: 0.0087, patient 6: 0.037, patient 7: 0.036). e , Average MFI of Lck on WT CD61 + , CD61 KO or WT CD61 + T cell clones (from patient 1), including on WT CD61 + T cell clone treated with 25 nM, 50 nM or 100 nM CD61 siRNA, or no treatment, by flow cytometry. f , Bar graph showing the average MFI of Lck on CD61 + T cell lines, following treatment with αCD61 neutralizing treatment, IgG isotype control treatment or no treatment, by flow cytometry. P values: (patient 1: 0.047, patient 2: 0.0059, patient 3: 0.011, patient 4: 0.049, patient 5: 0.048, patient 6: 0.0053, patient 7: 0.038). g , Representative histogram of phosphorylated Zap70 (pY292) on WT CD61 + T cell clone (from patient 1) following activation with or without Lck inhibition when using A770041 (right), or with genistein as positive control of tyrosine kinases inhibition (left), h , Schematic of CD61 modulation of Zap70 phosphorylation via Lck activity under no inhibition (left), after CD61 knock-down (middle) and Lck inhibition (right). Created with BioRender.com . d , f , n = 7 patients examined, three independent experiments. b , c , e , g , n = 3 independent experiments. a – f , Data are presented as the median ± s.e.m., *** P < 0.001, ** P < 0.01, * P < 0.05, one-way ANOVA with Tukey’s multiple-comparison test.

Article Snippet: In certain cases, cells were treated with 10 nM genistein (Santa Cruz Biotechnology) or 7.5 nM A770041 (Lck inhibitor, Sigma-Aldrich) before T cell activation.

Techniques: Clone Assay, Flow Cytometry, Activation Assay, Inhibition, Positive Control, Activity Assay, Comparison

Journal: Cell Reports Medicine

Article Title: CD28-CAR-T cell activation through FYN kinase signaling rather than LCK enhances therapeutic performance

doi: 10.1016/j.xcrm.2023.100917

Figure Lengend Snippet:

Article Snippet: LCK inhibitor A770041 , MedChemExpress , Cat# HY-11011.

Techniques: Purification, Functional Assay, Virus, Recombinant, Staining, Clone Assay, Mutagenesis, Enzyme-linked Immunosorbent Assay, Cytotoxicity Assay, Sequencing, Software