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. 2003 May 5;197(9):1221–1227. doi: 10.1084/jem.20022112

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Copyright © 2003, The Rockefeller University Press

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Figure 1. — Coaggregation of TCR and CD4 results in sequential activation of Lck then Fyn. (a) Total phosphotyrosyl content of each sample precoated with 1 μg/ml of biotinylated anti-TCRCβ (H57^b (1)) and/or 0.3 μg/ml of biotinylated anti-CD4 (GK1.5^b (0.3)) and coaggregated with 50 μg/ml of streptavidin for 10, 30, and 90 s, was assessed by immuno-blotting with phosphotyrosine specific mAb. The filter was stripped and successively probed with anti-Lck followed by anti-Fyn. (b) Cell lysates probed with pY394 Lck. The filter was stripped and probed with anti-Lck. (c) Fyn immunoprecipitates were probed with pY418 Src. The filter was stripped and probed with anti-Fyn. Histograms in b and c show the quantification of pY394 Lck and pY417 Fyn normalized to total kinase signals. The nonaggregated control sample was given a reference value “1.” All sample lanes in a, b, and c are aligned over a common legend.