Correlation of phosphorylation at Tyr-342 of FOXP3 with transcriptional regulation.

<p>(A) Comparison of the phosphorylation levels of FOXP3 mutants. The level of phosphorylated (top) and total (middle) FOXP3 immunoprecipitated and constitutively-active mutant of LCK (LCK CA) in cell lysates (bottom) was detected using Western blotting. Phosphorylation of Y330F and Y342F mutants of FOXP3 was greatly decreased compared with FOXP3 WT. (B) Comparison of MMP9 expression regulated by FOXP3 mutant. MMP9 expression was analyzed using a zymography assay (top). FOXP3, LCK CA, and actin expression was determined using Western blotting (middle). MMP9 expression level was normalized with actin (bottom). FOXP3 Y342F mutant was unable to suppress MMP9 unlike FOXP3 WT and Y330F mutant. The data represents the mean ± S.E. of three independent experiments. The asterisks indicate statistically significant differences (<i>p</i> < 0.01, Fisher's LSD test). (C) Real-time PCR analysis of <i>MMP9</i> in FOXP3 Y342F expressing cells. <i>MMP9</i> expression in FOXP3 Y342F cells was significantly increased compared with FOXP3 WT cells. The data represents the mean ± S.E. of three independent experiments. The asterisks indicate statistically significant differences (<i>p</i> < 0.01, Tukey-Kramer test). (D) <i>In </i><i>vitro</i> kinase assay. Analysis of phosphorylation of MBP-FOXP3 and MBP-FOXP3 Y342A (arrow) by LCK. Phosphorylation of MBP-FOXP3 (Y342A) was remarkably decreased compared with that of MBP-FOXP3 (WT). (E) Western blotting analysis using an anti-pTyr-342-specific antibody. FOXP3 and FOXP3 Y342F were immunoblotted with the anti-pTyr342 antibody. The antibody detected phosphorylation of FOXP3 only when LCK was co-transfected. (F) Levels of invasive cells. The cell number that invaded matrigel was normalized with cell counts that invaded the control insert. FOXP3 Y342F cells showed higher invasive rates than FOXP3 WT cells. The data represents the mean ± S.E. of six independent experiments. The asterisks indicate statistically significant difference (<i>p</i> < 0.01, Tukey-Kramer test).