Figure 4.

TCRxgag T cells exhibit deficiencies in Ras/MAPK and SAPK/JNK as well as in flux of Ca²⁺ after antigen stimulation. CD4-depleted splenocytes from TCR-TG and TCRxgag mice were stimulated with irradiated B6 splenocytes pulsed with 10 μg/ml gag-peptide. After stimulation, the cells were lysed in SDS-PAGE sample buffer at indicated time points. The samples were run on a SDS-PAGE gel and blotted. Blots were probed using primary Abs against phosphorylated forms of ERK1/2 (A) or JNK (B). Specific binding was detected by incubation with a secondary HRP-conjugated Ab and ECL Western blotting detection reagents. For analysis of Ca²⁺ flux (C), TCR-TG (black line) and TCRxgag (gray line) splenocytes expressing the Thy1.1 allomarker were depleted of CD4 cells and labeled with Indo-1. During the loading, cells were stained with Abs against Thy.1.1 and CD4. After washes, the cells were analyzed by flow cytometry in order to establish a base line. Control or peptide treated B6 Thy1.2 APC were added and after gating on Thy1.1⁺/CD4⁻ cells, the Ca²⁺ was followed over time. As a positive control, responder cells were treated with 1 μg/ml ionomycin.