Fig. 5

Immunoprecipitation of synthetic and endogenous A β by FCA antibodies

(A) Immunoprecipitations of synthetic Aβ40 and Aβ42 (1 µg) were performed in 5 ml of RIPA 1 × buffer with a 170-fold dilution of the IgG-purified fraction of the indicated FCAs. Immunoprecipitates were washed, electrophoresed on a 16.5% Tris-tricine gel, Western blotted, and probed with a 1:500 dilution of FCA18. Immunoreactivities were revealed with chloronaphtol as described in Materials and Methods. (B) Transfected HK293 cells overexpressing the Swedish mutated form of βAPP were obtained, cultured, and metabolically labeled as described in Materials and Methods. Conditioned media (10 ml) were collected, diluted in a one-tenth volume of RIPA 10 × buffer, incubated overnight with a 350-fold dilution of FCA18 (lanes 1), FCA3340 (lane 2), or FCA3542 (lane 3), then stirred for 4 hr in the presence of protein A-sepharose (100 mg/ml, 100 µl). Samples were centrifuged, washed three times with RIPA 1 × , resuspended with loading buffer, electrophoresed on a 16.5% Tris-tricine gel, and ra-dioautographed as described earlier (25). Arrow indicates the Aβ and p3 fragments.