Transport of A into cytosol through the plasma membrane was measured by ELISA and detected by confocal immunofluorescence and immunoelectron microscopies using anti-A IgG. Measurement of Phospho-MAPKs. S3 and and S3 and and and and and and and and and and SY-1365 are represented in Fig. S4 and and 0.001, versus WT; Unpaired oxidase (COX IV) activity in RAGE-deficient neurons as compared with COX IV activity in WT neurons. After exposure (24 h) to human A1C40 (Fig. 3 and and and and and and and 0.01, versus vehicle- and reversed A-treated cells (and 0.01, versus control (and and and and 0.05, **, 0.01, versus none (0 M A1C40) (and 0.01, versus control (no inhibitor). Membrane RAGE Acts as an A Carrier and SY-1365 Co-Internalizes with A. To determine molecular mechanisms underlying neuronal A transport, we biotinylated neuronal cell surface proteins, incubated the labeled cells with A1C40, and then analyzed internalized biotinylated proteins. First, we assessed the distribution of biotin in labeled cells before A treatment. Cells fixed immediately after biotinylation and permeabilized with detergent displayed a cell surface and focal [the latter SY-1365 were probably surface accumulations of biotin since they were removed by sodium 2-mercaptoethanesulfonate (MesNa) treatment; see below] distribution of the biotin (Fig. S9and and and and oxidase (COX IV) activity assays. Determination of Membrane A Transport. Transport of A into cytosol through the plasma membrane was measured by ELISA and detected by confocal immunofluorescence and immunoelectron microscopies using anti-A IgG. Measurement of Phospho-MAPKs. A-stimulated phosphorylation of SAPK/JNK and p38 MAPK was detected by Western blot analysis or measured by ELISA. Analysis of Internalization of Membrane Surface Proteins. Internalization of membrane surface proteins after A treatment was detected by Western blot analysis using biotinylation and immunoprecipitation. Immunohistochemistry. Immunohistochemistry was executed in hippocampal sections from Tg mAPP mice (9- to 10-month-old) and age- and strain-matched wild-type mice using anti-A IgG and SY-1365 anti-RAGE IgG. Statistics. Statistical analysis of the experimental data were carried out using GraphPad Prism 4 for Macintosh (GraphPad Software). The significance of differences was determined by a one-way ANOVA, SY-1365 followed by the Dunnett’s or Tukey’s multiple comparison test for multigroup comparisons. Unpaired TNFRSF10D 0.05. Supplementary Material Supporting Information: Click here to view. Acknowledgments. This study was supported in part by a grant for the 21st Century Centers of Excellence Program (1640102) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, a Grant-in-Aid for Scientific Research (18590050) from the Japan Society for the Promotion of Science, and a grant from Takeda Science Foundation. This work was also supported by grant from the U.S. Public Health Support (PO1AG17490). Footnotes Conflict of interest statement: D. Stern is usually a consultant for TransTech Pharma. This article is usually a PNAS Direct Submission. This article contains supporting information online at www.pnas.org/cgi/content/full/0905686106/DCSupplemental..