Varying activation conditions results in differential loss of CD3ζ.

Resting CD4 T cells (lane 1) were cultured for 72 hours in the presence of immobilized anti-CD3 (lane 2, anti-CD3), immobilized anti-CD3 and anti-CD28 (lane 3, CD3+CD28), or with anti-CD3 and autologous monocytes (lane 4, CD3+mc). Cells were purified through Ficoll, and residual monocytes were depleted with anti-CD14 coupled Dynabeads before lysis. Lysates containing equal protein levels (4.1 μg/ lane) were resolved on a 4% to 20% gradient reducing SDS-PAGE and blotted to nitrocellulose, cut into strips, and probed with anti–ZAP-70, antiactin, anti-CD3ε, and anti-CD3ζ antiserum. Lysates of purified monocytes (lane 5, mc) were run as blotting controls. The relative expression of CD3ζ determined by dividing the band densities in activated cells by the band density in resting CD4 T cells is indicated at the bottom of each lane. This partial reduction of CD3ζ expression with CD3 and CD3/CD28-stimulated T cells was observed in 5 different experiments.