NKG2D and TCR engagement induce cPLA₂ phosphorylation and AA release in CTLs. Celiac IE-CTL clone (A), PB-CTL clone (B), and TALL-104 CTLs (C) were studied. (left) Concentration-dependent cPLA₂ phosphorylation upon NKG2D and TCR engagement. CTLs were serum starved for 16 h, and then stimulated for 15 min as indicated. Cell lysates were analyzed by Western blot using anti–phospho-cPLA₂ (p-PLA₂)–specific antibody. The membrane was stripped and reblotted with anti-cPLA₂ antibody to assess equal loading. (right) AA release upon NKG2D and TCR engagement. CTLs were incubated with ³H-AA for 10 h, washed to remove free ³H-AA, and stimulated with the indicated antibody or isotype control for 1 h. Supernatants and cell pellets were separately harvested. Total and released ³H-AA were measured to determine the percentage of released ³H-AA. Mean value of triplicates was used for each of the three experiments. Data are means ± SD of three independent experiments. P values are from a Tukey-adjusted pairwise comparison.