Human phospho-Lck (Y394) Antibody (755103)

Detection of fmtslydn7u79t2logeav.jpg by Western Blot

Loss of phosphorylation at S224 and S241 prevents LAT tyrosine phosphorylation, binding to the signalosome, and cytokine production(A) JLAT transductants with DNA-PKcs phosphorylation sites 224 or 241 mutated, stimulated with 5 μg/mL alpha CD3/CD28 for 24 h, are unable to produce IL-2, as assessed by ELISA. Data are represented as mean ± SEM.(B) Western blot analysis of ZAP70 tyrosine phosphorylation events on LAT (Y132, 171, and 191) in resting or 2-min alpha CD3/CD28 (5 μg/mL) TCR-stimulated JLAT transductants. Following 2-min alpha CD3/CD28 stimulation, tyrosine’s 132, 171, and 191 are phosphorylated in WT and S224A JLAT transductants but not in S241A and S2A JLAT transductants.(C) ImageJ quantification of band intensity shows that WT and S224A LAT have tyrosine phosphorylation (132, 171, and 191) 9-fold higher than the S241 and S2A transductants, with one dot representing one experiment. Data are represented as mean ± SEM.(D) ZAP70 tyrosine phosphorylation of residues 132, 171, and 191 on LAT is blocked with pan-PI3K inhibitor LY294002 (20 μM) but not with selective PI3K inhibitors in WT LAT transduced JLAT T cells.(E) ImageJ quantification of band intensity shows tyrosine phosphorylation (132, 171, and 191) 2.5-fold lower in WT LAT-transduced JLAT T cells treated with LY294002 compared to other inhibitors tested, with one dot representing one experiment. Data are represented as mean ± SEM.Representative blots from n = 3 independent experiments. One-factor ANOVA plus Tukey’s multiple comparisons to determine statistical significance ( alpha = 0.05, *p < 0.05, **p < 0.01, ***p < 0.0005, and ****p < 0.0001). Image collected and cropped by CiteAb from the following open publication (https://linkinghub.elsevier.com/retrieve/pii/S2211124725015682), licensed under a CC-BY license. Not internally tested by R&D Systems.