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. 2019 Jun 12;9:8524. doi: 10.1038/s41598-019-44317-w

Figure 6.

Figure 6

PrPC but not PrPA116V promotes secretion of Aβ from N2aAPPswe cells. (A) Western blot confirms PrPC knockdown and PrPA116V expression in N2aAPPswe cells used in Aβ secretion studies. Cells were transfected with a non-silencing control siRNA as control (CTL) (lanes 1 and 2), or they were transfected with anti-PrPC siRNA (lanes 2 and 3), or co-transfected with anti-PrPC siRNA and a pCB6 expression vector containing PrPA116V (lanes 5 and 6). The transfection media was removed 24 h later and the cells were incubated with OPTI-MEM I for 24 h, then lysed and harvested for Western blotting. From cell lysates, 30 μg of protein was subjected to 12.5% SDS-PAGE then transferred to PVDF membranes and probed with anti-PrP SAF-32 mAb and α-tubulin. (B) N2aAPPswe cells were grown on coverslips and transfected as in A, then prepared for immunofluorescence and labeled with anti-PrP mAb SAF-32 and anti-Aβ42 antibody PA3-16761. Confocal fluorescence images of cells are shown at 20X (left panel) and 100X (right panel) magnification. Scale bars = 10 μm. (C) Bar graph displays ELISA measurements (pg) of total Aβ from control N2aAPPswe cells (CTL), after PrP knock down (PrP−/−), and after PrP knock down combined with PrPA116V transfection. Samples used were 30 μg of protein from lysates and 5 μL of 10 mL media. Total human Aβ42 levels were calculated from the measures of total lysate and total media. Six samples per group with 2 replicates per sample were tested. See Supplemental Table 5 for actual values. ANOVA p > 0.05. (D) Intracellular (solid bars) and secreted (open bars) Aβ (pg) measured by ELISA from a fraction of cell lysate and media, respectively. Six samples per group with 2 replicates each were tested. See Supplemental Table 5 for actual values. ANOVA results for intracellular Aβ in cell lysates: p < 0.01, post hoc multiple comparisons test, **p < 0.01 between CTL and PrP−, CTL and PrPA116V; p > 0.05 between PrP− and PrPA116V cells. Aβ in media, p < 0.01 ANOVA, post hoc multiple comparisons test, **p < 0.01 between CTL and PrP−, and PrPA116V; p > 0.05 between PrP− and PrPA116V cells.