*and and and and and at 4C (Beckman). (Abcam, 1250), mouse Brucine monoclonal anti-AT1R, which recognizes the AT1R carboxyl terminal (Abcam, 1125), and mouse monoclonal anti- actin (Sigma, 11,000). Equivalent amounts of protein components (about 60 g) were electrophoresed in sodium dodecyl sulfate-polyacrylamide gels (Bio-Rad). Proteins were transferred onto polyvinylidene difluoride membranes (Millipore). Non-specific binding was clogged with 5% non-fat dried milk in 50 mmol/l Tris-HCL, pH 7.4, containing 200 mmol/l sodium chloride and 0.5 mmol/L Tween 20 (Tris-buffered saline Tween). The blots were incubated with different main antibodies, followed by incubation with peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulins in Tris-buffered saline Tween comprising 10% nonfat dried milk. Reactions were developed with an enhanced chemiluminescence system, according to the manufacturer’s protocols (Pierce). The blots were then exposed to films (Kodak) for numerous periods of time (0.05C5 min). Data were normalized to the -actin band. Fluorogenic substrate assay Assays to evaluate secretase activity were performed relating to a recent study [89]. Briefly, rat cerebral cortices were isolated and homogenized. The producing aliquots (comprising 15 g of proteins) were centrifuged at Mlst8 12,000 for 10 min (Beckman). The membrane-enriched fractions were then incubated at 37C for 30 min in 50 l assay reaction buffer (for -secretase, 10 mmol/L Tris-HCL, pH 6.8; for -secretase, 50 mmol/l sodium acetate, pH 4.5; for -secretase, 50 mmol/l Tris-HCL, pH 6.8, 2 mmol/l EDTA, 0.25% CHAPSO) containing 10 mol/l fluorescent conjugated peptide substrate (Calbiochem) at 37C for 2 hr. The degree of substrate cleavage Brucine was measured as the emitted fluorescence using a reader (Tecan) at excitation/emission wavelengths of 325/393 nm (for -secretase), 320/420 nm (for -secretase) and 355/440 nm (for -secretase). Enzyme-linked immunosorbent assay (ELISA) The levels of soluble A40 and A42 in rat cerebral cortices were recognized using sandwich ELISA packages (Wako), according to the manufacturer’s instructions. All samples were analyzed in duplicate. Statistical analysis Data were analyzed using analysis of variance (ANOVA), followed by least significant difference checks, using SPSS 13.0 software (SPSS). All results were indicated as meanstandard Brucine deviation (SD). Assisting Info Number S1 Concentration assays for A40 and A42 by ELISA. n = 6 for each group. All samples were analyzed in duplicate. * em p /em 0.05 or ** em p /em 0.01 versus the control group receiving saline infusion. (TIF) Click here for more data file.(118K, tif) Footnotes Competing Interests: The authors possess declared that no competing interests exist. Funding: This study is supported by Key Project of Medical Technology and Technology Development Foundation, Nanjing Division of Health (NO.ZKX08035, http://www.njh.gov.cn/html/list_83.shtml). The funders experienced no Brucine part in study design, data collection and analysis, decision to publish, or preparation of the manuscript..