Fig 1: G3P directly interacts with LCK and suppresses its activity. a The p-LCK(Y394) levels of human CD8+T cells treated with gradient concentrations of G3P. b Immunoblot analysis of the indicated proteins in human CD8+T cells treated with gradient concentrations of G3P. c In vitro kinase assay using purified LCK and its mutants incubated with ATP and increasing amounts of G3P. d Purified LCK protein was analyzed by SDS–PAGE, followed by Coomassie blue staining. e BIAcore measurement of the interaction between G3P and the purified LCK. f The interaction between LCK and G3P were speculated using three-dimensional structures. g Schematic of LCK structure. h Purified region LCK (60-240AAs) was analyzed by SDS–PAGE, followed by Coomassie blue staining. i BIAcore measurement of the interaction between G3P and the purified region. j Purified region LCK(240-509AAs) was analyzed by SDS–PAGE, followed by Coomassie blue staining. k BIAcore measurement of the interaction between G3P and the purified region. l Purified region LCK(230-390AAs) was analyzed by SDS–PAGE, followed by Coomassie blue staining. m BIAcore measurement of the interaction between G3P and the purified region. n Purified region LCK(330-509AAs) was analyzed by SDS–PAGE, followed by Coomassie blue staining. o BIAcore measurement of the interaction between G3P and the purified region
Fig 2: G3P inhibits CD8+T cells through LCK signaling. a The TCR signaling pathway. b CD8+T cells co-cultured with shCtrl or shVcp Hepa1-6-OVA cells were activated for 5 or 10 min or unstimulated (0). Cell lysates were analyzed by western blot (WB). c Mouse naive CD8+T cells treated with G3P or untreated (Ctrl) were activated for the indicated times. Cell lysates were analyzed by western blot (WB). d–g Left, mouse naive CD8+T cells treated with G3P or untreated (Ctrl) followed by stimulation were subjected to immunostaining and confocal microscopic imaging. Right, quantification of the mean fluorescence intensity. h Immunoblot analysis of the indicated proteins in mouse CD8+T cells treated with G3P and/or PP2 during stimulation. i The expression of activation indicators in mouse CD8+T cells treated with G3P and/or PP2 was determined by flow cytometry (n = 3). j The percentage of cytokines produced by mouse CD8+T cells treated with G3P and/or PP2 was measured by flow cytometry (n = 3). k Jurkat T cells expressing WT–LCK or the LCK(Y394F) mutant were treated with G3P or untreated. The expression of activation indicators was determined by flow cytometry (n = 3). Data are presented as mean values ± SD. Statistical significance was determined using two-sided t-tests, *P < 0.05, **P < 0.01, and ***P < 0.001
Supplier Page from Abcam for Phospho-Lck (Y394) ELISA Kit