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. Author manuscript; available in PMC: 2010 Oct 1.
Published in final edited form as: Clin Immunol. 2009 Oct;133(1):145–153. doi: 10.1016/j.clim.2009.06.006

Figure 2. Activation of ZAP-70 and Syk by A2 and A9.

Figure 2

A) The qCII24 CD4+ T cells were activated by APCs pre-pulsed with 100 μg/ml of A2, A9 or no peptide (No Ag) for 5 minutes. This time point was selected based on a time course analysis showing that the peak activation occurred between two to five minutes after stimulation. Whole cell lysates were collected and the proteins were separated by SDS-PAGE. The proteins weretransferred onto PVDF membranes and analyzed first using antibodies specific for ZAP-70 (pZAP-70-y493) and Syk (pSyk-y525). The membrane was then stripped and reprobed with antibodies to total ZAP-70 (ZAP-70) and total Syk (Syk) respectively. B). The T cells were stimulated by APCs pre-pulsed with 100 μg/ml of A2, A9 or no peptide (Control) for 2 minutes. The cells were then fixed, permeabilized, stained with an antibody specific for phospho-ZAP-70 (top panel) or an antibody specific for phospho-Syk (bottom panel) and analyzed by flow cytometry. The histograms were gated on CD4+ T cells.