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. 2014 May 27;5:3867. doi: 10.1038/ncomms4867

Figure 3. Impact of scFvA13-KDEL in 7PA2 cells on the extracellular patterns of Aβ species.

Figure 3

(a) WB analysis of secreted AβOs in the CM of 7PA2 cells and of 7PA2 stably expressing the scFvA13-KDEL intrabody (7PA2-A13K). LDS/SDS-stable Aβ dimers and Aβ trimers were detected by WB using two different anti-Aβ mAbs, mAb 4G8 (against a.a. 17–25 of Aβ) and mAb 6E10 (against a.a. 3–9 of Aβ), as primary antibodies (see also Supplementary Fig. 5). Aβ dimers and Aβ trimers are significantly reduced in 7PA2-A13K, whereas no significant changes in the 4 kDa Aβ bands (deriving both from monomers and LDS/SDS unstable oligomers) are observed (see also Supplementary Figs. 3,5). The histogram shows the levels of Aβ species in 7PA2-A13K CM in comparison with 7PA2 CM (at 100) obtained from densitometric values of bands and normalized as discussed in the method section and in the Supplementary Fig. 5. Mean values±s.e.m., n=4, *P<0.01, Student’s t-test. (b) DB analysis of AβOs in CM were done by using the conformation-sensitive and sequence-specific scFvA13 or scFvIm3 anti-AβOs13 as primary antibodies (and detected through anti-His tag). The levels of AβOs are significantly decreased in 7PA2-A13K CM, in comparison with 7PA2 CM, similarly to those of Aβ dimers and Aβ trimers (measured by WB). The histogram shows the levels of AβOs in 7PA2-A13K CM in comparison with 7PA2 CM (at 100) obtained from densitometric values of dots and normalized as discussed in the method section. Mean values±s.e.m., n=4, *P<0.01, Student’s t-test. (c) Immunoproteomic analysis of secreted Aβ monomers and fragments. The levels of N- and C-terminally truncated Aβ monomers, measured by 6E10+4G8 SELDI-TOF MS, are significantly increased in 7PA2-A13K CM, in comparison with 7PA2 CM (all ratios in Supplementary Table 2). The most abundant Aβ species are increased by 53.1±1.6% (Aβ1-37), 44.2±1.1% (Aβ1-38), 48.6±0.9% (Aβ1-39), 50±0.3% (Aβ1-40), 59±3.3% (Aβ1-42), respectively. The histogram shows the intensity (microampere, uA) of single proteomic peaks. Mean values±s.e.m., n=4, P<0.001, Student’s t-test.