Figure 2

A) HeLa cells were transfected with pcDNA3 (-) or CD74 (+), together with Notch. The total cell lysates (TL) and FLAG IPs were analyzed as in Figure 1B. Notch was detected with αmyc antibody. B) HeLa cells were transfected with pcDNA3 (-) or CD74 (+), together with APLP2, and were analyzed as in A. C) Left panels: HEK293APP cells were transfected with the indicated plasmids. The transfected cells were further incubated for 24 hours with fresh media, and secreted sAPPα/β were measured by Westerm blot. The cell lysates were analyzed as in Figure 1B. Right panels: the quantification of sAPPα/β. The levels in pcDNA3 transfected cells were set to 100. D) HEK293APP cells were transfected with pcDNA3 or CD74 and treated as in C. Aβ40 and 42 secreted in the media, and Aβ40 in the lysates were measured with specific ELISA. The amount of Aβ was normalized to the amount of the protein in the total lysates. The asterisks (**) indicate that p values by Student's t test are less than 0.01. E) HeLa cells were transfected with pcDNA3 or CD74, and the media was replaced with fresh media containing synthetic Aβ40 and Aβ42. The media of pcDNA3 (open circles) and CD74 (closed circles) transfected cells were incubated for indicated hours, and the remaining Aβ40 and Aβ42 were measured as in D. F) HeLa cells were transfected with pcDNA3 or FLAG-CD74 together with APP. The cells were pulsed for 30 min with ³⁵S methionine+cysteine, and chased in fresh media for indicated hours. APP was immunoprecipitated with αAPPct antibody and analyzed by SDS-PAGE followed by autoradiography. G) HeLa cells were transfected with pcDNA3 (-) or FLAG-CD74 (+) together with wild type APP (wt APP) or the indicated mutants. The total lysates and FLAG IPs were processed as in Figure 1B. Note that APP Ncas lacks C-terminal 31 amino acids of APP and has therefore a smaller in size.