Fig. 6

Phosphorylation of Zap70 selectively requires iron supply via transferrin-TfR system. A–E Immunoblots with phosphorylation-specific antibodies against proteins involved in proximal TCR signalling. Jurkat T cell activation was accomplished with soluble anti-CD3ε + anti-CD28 for the indicated times before cell lysis. Data from 4–5 independent experiments. A After blotting, the nitrocellulose membrane was probed with anti-phospho-Zap70 (Y319). Beta actin was used as loading control. Left: Band intensity quantification. Right: Relevant parts of one representative immunoblot. B Membrane was probed with anti-phospho-LAT (Y220). After membrane stripping, beta actin was used as loading control. Left: Quantification of band intensities. Right: Relevant parts of one representative immunoblot. C After blotting, the nitrocellulose membrane was probed with anti-phospho-SLP-76 (Y145). Beta actin was used as loading control. D Membrane was probed with anti-phospho-Vav1 (Y174). Beta actin was used as loading control. E After blotting, the nitrocellulose membrane was probed with anti-phospho-Src family (Y416). Beta actin was used as loading control. F Primary T cell activation was accomplished with soluble anti-CD3ε + anti-CD28 for the indicated times before cell lysis and SDS PAGE. After blotting, the nitrocellulose membrane was probed with anti-phospho-Zap70 (Y319). Beta actin was used as loading control. Left: Quantification of reduction in Zap70 phosphorylation. Right: Relevant parts of one representative immunoblot. Error bars indicate mean ± SEM. Statistical significance determined with two-way ANOVA (A–E) or one-sample t-test (F). *p < 0.05; **p ≤ 0.01; no indication—not significant