Figure 6 | Cell Death & Disease

Figure 6

From: TGF-β induces TIAF1 self-aggregation via type II receptor-independent signaling that leads to generation of amyloid β plaques in Alzheimer's disease

Figure 6

TIAF1 aggregation in vivo and generation of Aβ in vitro. (a) Water-soluble TIAF1 protein aggregates were analyzed by non-reducing SDS-PAGE (5%) and western blots using the lysates of hippocampal samples from postmortem AD patients and nondemented controls. The blots were stained with specific anti-TIAF1 antibody (also see Supplementary Figure S13). C, control; A, Alzheimer's disease. (b) Filter retardation assay shows the presence of ‘water-insoluble’ TIAF1 aggregates in control and AD hippocampi (representative data from 138 samples). The amounts of protein loaded were 10, 30 and 60 μg, respectively, from top to bottom. (c) TIAF1 aggregation is shown in 59% of nondemented control hippocampi (age 59.0±17.0, n=41), and only 15% of the total samples have Aβ aggregates. In comparison, 54% of TIAF1 aggregation is shown in the hippocampi of older postmortem AD patients (80.0±8.8, n=97), and 48% of the total AD samples possess Aβ aggregates. In addition, there was 17% of the total TIAF1-positive samples possessing Aβ aggregates in the control group, and 48% in the AD group. (d) L929 cells were treated with TNF-α (50 ng/ml) for 24 h. Presence of Aβ monomer (∼4.5 kDa) is weakly shown in the whole cell lysate (see load), and the band was enriched by immunoprecipitation using anti-Aβ antibody (AβH43). (e and f) Transiently overexpressed ECFP-TIAF1/EYFP-TIAF1 (Tc/Ty) induced generation of amyloid fibrils (dot blot; AO antibody), AICD and Aβ (reducing SDS-PAGE), but not in cells overexpressing ECFP/EYFP (C/Y) or ECFP-TIAF1/mutant EYFP-TIAF1 (Tc/mTy). TGF-β1 (5 ng/ml) induced the expression of prefibril Aβ oligomers (A11 antibody) in C/Y-expressing L929 cells. Mutant or dominant-negative TIAF1 is with an E22/23A alteration. (g) Similar experiments were performed in Mv1Lu cells, which shows Tc/Ty-induced amyloid fibrils (a single band under reducing SDS-PAGE; ∼60 kDa). (h and i) NCI-H1299, B16F10 and SH-SY5Y cells were treated with medium C, TGF-β1 (5 ng/ml), -β2 (5 ng/ml), -β3 (5 ng/ml), TNF-α (50 ng/ml) and TNF-β (25 ng/ml) for 24 h. Distinct APP degradation patterns are shown. A representative protein gel is shown from three repeats e–i

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