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. 2001 May 22;98(11):6336–6341. doi: 10.1073/pnas.101133498

Figure 2.

Figure 2

Effect of pHN and secretion of the transcribed HN into cultured media. (A and B) F11 cells were transfected with pcDNA or either V642I-APP, M146L-PS1, or N141I-PS2 cDNA with pFLAG or pHN for 72 h. In A and B, cell mortality and viability were measured, respectively. Negative controls without transfection (no T) or with empty plasmid transfection (vec) were also examined. (A Insets) Expression of cognate FAD proteins by transfection with FAD genes. Arrows indicate the cognate holoproteins (1, no T; 2, vec; 3, FAD gene; 4, FAD gene + pHN). (C) F11 cells were transfected with pcDNA or V642I-APP cDNA in the absence of serum for 3 h, incubated with HF-18% for 2 h, and cultured with CM/pHN, other transfected CM, or fresh media (fresh HF-10%) for 67 h. Cell mortality was measured 72 h after transfection. (D) F11 cells were transfected with pHN, pS7A (pHN mutant with S7A), or pHNR for 72 h, and the cell lysates (Lower) and media (Upper) were analyzed by M2 antibody. (E) F11 cells were transfected with pHN for 24 h and treated with or without secretagogues or an inhibitor for 6 h. CM and cell lysates were submitted to immunoblot analysis with M2 antibody [1, no transfection; 2, pHN transfection alone; 3, pHN + 48 mM KCl; 4, pHN + 1 μM forskolin; 5, pHN + 0.1 mM 3-isobutyl-1-methylxanthine (IBMX); 6, pHN + 1 μM forskolin + 0.1 mM IBMX; 7, pHN + 20 ng/ml (+)Brefeldin A]. (F) F11 cells were transfected with pcDNA or V642I-APP cDNA with cotransfection of pFLAG or pHN or with treatment of 10 μM HN peptides. After 72-h culture, cell mortality was measured. *, significant suppression (P < 0.01); **, no significant suppression vs. V642I-APP-induced cell death.