Fig. 2
From: Tau seeding activity in various regions of down syndrome brain assessed by two novel assays
Seeded-tau aggregation assay in cultured cells to evaluate the seeding activity of AD O-tau. a AD O-tau seeded tau151-391 aggregation in HeLa cells. HeLa cells were transfected with pCI/HA-tau1-441 or pCI/HA-tau151-391, treated with AD O-tau for 42 h after 6 h tau transfection, and immunostained with anti-HA. b AD O-tau seeded tau151-391 aggregation in HEK-293FT cells. HEK-293FT cells were transfected with pCI/HA-tau1-441 or pCI/HA-tau151-391 and treated with AD O-tau, as described in panel A. The cells were lysed with RIPA buffer. RIPA-insoluble and -soluble fractions yielded by ultracentrifugation were analyzed with Western blots developed with anti-HA. c AD O-tau induced tau151-391 aggregation dose-dependently in cultured cells. HEK-293FT cells overexpressing HA-tau151-391 were treated with various amounts of AD O-tau for 42 h. The RIPA-soluble and -insoluble tau were analyzed by Western blots. The levels of RIPA-insoluble tau were plotted against various amounts of AD O-tau. d Phosphorylation of RIPA-insoluble tau was different from that of sarkosyl-insoluble tau. HEK-293FT cells overexpressing HA-tau151-391 were treated with AD O-tau. The cells were lysed with RIPA-buffer or 1% Sarkosyl buffer. The insoluble and soluble tau were analyzed by Western blots developed with anti-HA or with anti-phospho-tau (pT205- or pT231-tau). e, f AD O-tau seeded 3R-tau151-391, but not TDP-43, aggregation. HEK-293FT cells expressing HA-3R-tau151-391 or HA-TDP-43100–414 were treated with various amounts of AD O-tau for 42 h. RIPA-insoluble and -soluble 3R-tau151-391 (e) or TDP-43100–414 (f) were analyzed by Western blots developed with anti-3R-tau (RD3) or anti-HA