Src-family kinases negatively regulate NFAT signaling in resting human T cells
Fig 4
Src-kinase Lck prevents aberrant NFAT1 activation in unstimulated Jurkat T cells.
(A) Immunoblot analysis of Lck expression in Jurkat T cells either expressing (Lck+) or not expressing (Lck-) Src-kinase Lck. GAPDH served as a loading control. (B) NFAT1-dependent firefly luciferase activity (NFAT1-Luc) was measured in Lck+ or Lck- Jurkat T cells. Firefly luciferase activity was normalized to renilla luciferase activity. (C) Immunoblot analysis of subcellular localization of NFAT1, GAPDH and Lamin in Lck+ or Lck- Jurkat T cells. GAPDH and Lamin served as a loading control for cytoplasmic and nuclear fractions respectively. NFAT1 protein levels in the cytoplasmic fraction and nuclear fractions were normalized with GAPDH and Lamin protein levels respectively. (D) Immunoblot analysis of NFAT in nuclear fraction obtained from Lck-deficient (Lck-) and Lck expression rescued (Lck-/Lck) Jurkat T cells. Activation of distal TCR signaling was measured by assessing (E) IL-2 release and (F) CD25 surface expression in Lck+ Jurkat T cells following treatment with DMSO or Lck-specific inhibitor (Lck inhibitor II) and PMA/Ionomycin stimulation. RLU = relative luciferase units. Data represent the average of three technical replicates, and the standard deviation is shown. Each experiment was independently performed three times with similar results. *P< 0.01.