Fig. 4: Methylation at K668 determines NFAT5 nuclear localization and activation.
From: Lysine methylation promotes NFAT5 activation and determines temozolomide efficacy in glioblastoma
a Identification of lysine methylation site of NFAT5 by LC-MS/MS, corresponding to one of two methylation sites, K668. b K668 (but not K616) mitigated EGF-induced NFAT5 tri-methylation and EZH2/NFAT5 interaction. c Knockdown of EZH2 by shRNA or DZNep mitigated EGF-induced NFAT5 K668 methylation in U87/EGFR cells. d Expression of Me3-NFAT5 K668 and the association between NFAT5 and EZH2 was analyzed in HA-tagged EZH2 WT, S21A, S21D expressing U251 cells. e IF staining the subcellular ___location of NFAT5 in U251 cells stably expressing NFAT5 WT or K668R mutant. Scale bar: 20μm. One representative field of n = 30 independent cells was captured. f The nuclear and cytosolic fractions of U87/EGFR cells expressing NFAT5 WT or K668R mutant treated with EGF at different time were subjected to immunoblotting analysis. g Co-IP assay was performed to examine IMB1 as an interactor of NFAT5 in the U87/EGFR and U251 cells. h The association of NFAT5 and IMB1 was examined in NFAT5 WT or K668R U87/EGFR cells in the presence of EGF or not. i The nuclear and cytosolic expression of NFAT5 and Me3-NFAT5 K668 was detected in EZH2 overexpressing cells transfected with shNC or shIMB1. j NFAT5 TAD reporter luciferase activity was measured in cells transfected with NFAT5 WT or K668R. (b–j) n = 3 independent experiments. Significance was calculated by (j) one-way ANOVA with LSD-t. Data were presented as mean ± standard deviation. Marker unit for Western blots is kDa. Source data are provided as a Source Data file.
