FIGURE 2:

DGK recruitment to active TCR complex; DGKζ is necessary for PA production. (A) Jurkat T cells were presented to anti-CD3/CD28–coated Dynabeads for various times. Cell treatment and lysate precipitation were as in Figure 1B. TCR immune complexes were eluted (TCR bound), resolved by SDS–PAGE, and probed in WB with DGK- and activation marker–specific antibodies. Unprecipitated samples were used as activation controls (total cell lysate [TCL]). (B, C) DGKζ is the main PA-generating enzyme recruited to the TCR complex. RNAi was used to silence DGKα and DGKζ; at 72 h posttreatment, cells (10⁷ cells/ml) were starved as in Figure 1B and stimulated with anti-CD3/CD28 beads for various times. TCR-bound samples or TCL (input, I) were used as an enzyme source for the DAG kinase assay (type I; see Materials and Methods); a representative experiment is shown (B). PA production is shown as the mean ± SEM (n = 4). A fraction of the samples used in B was analyzed by WB to confirm RNAi efficiency and activation (C).