Table 2.
Differences in the proximity between N and C termini in the PS1 holoprotein and wild-type heterodimer
|
Condition |
FITC lifetime (t2; mean ± SD) |
p value (compared with t1, FITC-IgG, 2321 ± 59) |
|---|---|---|
| PS1 wild type (n = 55) | 1768 ± 141 ps | <0.001 |
| PS1 D257A (n = 38) | 2450 ± 126 ps | ns |
| PS1 D385A (n = 12) | 2389 ± 82 ps | ns |
| PS1 (Nct RNAi) (n = 28) |
2199 ± 185 ps |
ns |
FITC lifetime (t1) in the absence of FRET (in PS1 NT-BiP double-immunostained cells; negative control) was calculated using FLIM software. FITC-IgG lifetime (t1) was 2321 ± 59 ps. To analyze PS1 NT and CT proximity, the average FITC lifetimes per cell were fitted to two curves representing the non-FRETing (fixed as t1) and FRETing (t2) populations. These calculated lifetimes were recorded per cell and are summarized. A t2 lifetime shorter than t1 lifetime implies the presence of FRET and a close proximity between the donor and acceptor. To generate PS1 holoprotein, the cells were transfected with either Nct RNAi 21-mer dsRNA or D257A, or D385A PS1-expressing stable cell lines were used. One-way ANOVA was performed to analyze differences in the lifetime, followed by least significant difference post hoc analysis. Levene's test was also performed to determine whether variances were equal. n, Cell number; ns, not significant (ANOVA).