FIG. 3.

Proximal TCR signals are defective in the absence of Lck. CD4 T cells were stimulated with anti-CD3, anti-CD3 plus anti-CD4, or anti-CD3 plus anti-Thy1, as shown, for 2 min. Total cell lysates were run on SDS-PAGE gels, immunoblotted for TCRζ chain phosphorylation with anti-pY and reprobed for TCR-ζ (A), and immunoblotted for pY319-ZAP-70 (pZAP) and total ZAP-70 (tZAP) (B and C). (D) ZAP-70 kinase activity from ZAP immunoprecipitates was measured by ³²P incorporation in vitro on GST-LAT substrate. Total ZAP-70 indicates loading. (E) Immunoblots probed with phospho-specific LAT Abs for pY136, pY175, pY195, and pY235 and those shown in panels A to D were visualized with chemiluminescence. (F) Graphs quantifying individual phospho-LAT residues either from the densitometries of ECL blots or directly from a Li-Cor Odyssey infrared imager for three to five replicate experiments are normalized to data for their loading controls and expressed as percentages of the maximum stimulation level, set to 100% based on WT cells stimulated with CD3 plus CD4. (G) T cells expressing Lck plus Fyn (Lck^ON), Fyn only (Lck^OFF), Lck only (Lck^ON Fyn^−/−), or neither kinase (Lck^OFF Fyn^−/−) were stimulated as described above and lysates probed for pLAT 175 and total LAT to control for loading and visualized with a Li-Cor infrared imager.