Figure 5.

Analysis of the action of HN. (A) F11 cells were incubated with radiolabeled sHNG in the presence or absence (1) of 2 μM unlabeled sHNG (2) or sHNA (3) for 2 h at 4°C and then treated with 200 μM BS³ for 20 min at 4°C. After adding Tris⋅HCl buffer and washing cells with PBS four times, cells were resuspended in PBS and subjected to radiocounting. The total input of radioactivity was 590,990 cpm/sample. (B) F11 cells were transfected with pcDNA or V642I-APP cDNA and incubated with or without 10 μM sHN in the presence or absence of 10 nM wortmannin (W), 100 μM genistein (G), or 50 μM PD98059 (PD). Seventy-two hours after the onset of transfection, cell mortality was similarly measured. (Inset) Jurkat cells were incubated with or without 100 ng/ml CH-11 (anti-Fas; MBL) in the presence or absence of 10 μM sHN (anti-Fas + sHN) for various periods, and cell mortality was similarly measured. (C) F11 cells were transfected with CN-procaspase-3 cDNA with pcDNA or V642I-APP cDNA by lipofection [0.2 μg CN-procaspase-3 cDNA, 0.8 μg V642I-APP cDNA, LipofectAMINE 2 μl, PLUS Reagent 4 μl (Upper); 0.4 μg CN-procaspase-3 cDNA, 0.6 μg V642I-APP cDNA, LipofectAMINE 2 μl, PLUS Reagent 4 μl (Lower)] in the presence or absence of 10 μM sHN or 100 μM Ac-DEVD-CHO [no transfection (lanes 1, 6); pcDNA transfection (lanes 2, 7); CN-procaspase-3 transfection (lanes 3, 8); CN-procaspase-3 + V642I-APP (lanes 4, 9); CN-procaspase-3 + V642I-APP + sHN (lanes 5, 10); CN-procaspase-3 + V642I-APP + Ac-DEVD-CHO (lane 11)]. Forty-eight hours after onset of transfection, cell lysates were submitted to immunoblot analysis with anti-caspase-3, anti-APP, or anti-tubulin antibody. Arrows, procaspase-3.