Fig. 6.
Activation of human PMNs by PAF triggers rapid mTOR-dependent phosphorylation of S6K1 and 4E-BP1. (A) Freshly isolated human PMNs were lysed immediately (control; lane 1) or were activated with 10 nM PAF (lane 2) and the mobility of S6K1 was assayed by immunoblotting. In parallel, PMNs were pretreated with rapamycin and activated with PAF for the times shown (lanes 3–7). (B) Freshly isolated PMNs were pretreated with control buffer (lanes 1–4), rapamycin (lanes 5–8), or FK506 (lanes 9–12) and activated with PAF for the times shown followed by immunoblotting. More slowly migrating bands represent phosphorylated S6K1, as in A. (C) Freshly isolated human PMNs were lysed immediately (control; lane 1) or after activation with 10 nM PAF for the times shown after pretreatment with control buffer (lanes 2–5) or rapamycin (lanes 6–10). The lysates were then probed for 4E-BP1 by using a phosphospecific antibody that recognizes phosphorylation on serine-65, a critical residue (20).