Upon sucrose density gradient fractionation, lipid rafts were recovered mainly in fraction 4, where flotillin-1 was predominantly present. A proportion of BACE1 was recovered in fraction 4, while the majority was present

in high-density nonraft Wortmannin cost fractions (fraction 8–10) (Fig. 4a). The percentage of BACE1 in the raft fraction was 14% and 3% in neurons expressing BACE1-WT and BACE1-CA4, respectively (Fig. 4b), confirming that lipid raft association of BACE1 is dependent on its palmitoylation in primary neurons as well. Figure 4 Lipid raft Inhibitors,research,lifescience,medical distribution of BACE1 depends on palmitoylation in primary rat cerebral cortical Inhibitors,research,lifescience,medical neurons. (a) CHAPS extracts of cultured neurons infected with recombinant adenoviruses expressing BACE1-WT or BACE1-CA4 were fractionated via sucrose density gradient … A proportion of APP was detected in fraction 4, with the majority recovered from nonraft fractions. The percentages of APP in the raft fraction were similar for SH-BACE1-WT- and SH-BACE1-CA4-expressing cells (Fig. 4c), suggesting that raft distribution of BACE1 does not directly affect that of APP. Inhibitors,research,lifescience,medical We additionally evaluated the cellular localization of BACE1 using double immunofluorescence staining. Immunostaining with 1D4 revealed localization

of BACE1 in both soma and neurites. There were no appreciable differences in the staining Inhibitors,research,lifescience,medical patterns between BACE1-WT- and BACE1-CA4 (Fig. 4d). Upon double labeling with 1D4 and anti-flotillin-1, flotillin-1 immunoreactivity was observed as punctate staining that partially overlapped that of 1D4. The extent of the colocalization of 1D4 and flotillin-1 immunoreactivities appeared reduced in Inhibitors,research,lifescience,medical neurons expressing BACE1-CA4, relative to those expressing BACE1-WT (Fig. 4d), consistent with the results of biochemical fractionation. Aβ production is not influenced by raft association of BACE1 in neurons Since lipid rafts appear

to represent an important site for amyloidogenic processing of APP by BACE1 (Cordy et al. 2006; Araki 2010; Rushworth and Hooper 2010; Vetrivel and Thinakaran 2010), Megestrol Acetate we analyzed the secretion of Aβ from primary neurons overexpressing BACE1-WT or BACE1-CA4. On Western blots, neurons expressed comparable levels of BACE1-WT and BACE1-CA4 (Fig. 5a). BACE1-WT and BACE1-CA4 enhanced secretion of both Aβ40 and Aβ42 to similar extents (by approximately 80%), compared to control cells infected with empty adenovirus (Fig. 5b and c). Figure 5 Neuronal Aβ production is not influenced by raft association of BACE1. (a–c) Cultured neurons were infected with recombinant adenoviruses expressing BACE1-WT, BACE1-CA4, or empty adenoviruses (mock). (a) Cell lysates were analyzed by Western …