Las 3000 plus

Kidney and liver tissues were homogenized in lysis buffer containing 300 mM NaCl, 50 mM Tris–HCl, 0.5% Triton X-100, and protease-inhibitor cocktail (pH 7.6). The incubation was performed at 4 °C for 30 min. After centrifugation at 14,000 rpm for 20 min at 4 °C, protein concentrations of the lysates were determined with the Bradford protein assay reagent (Bio-Rad, Hercules, CA). The proteins (40 μg) were loaded into 7.5–15% SDS/PAGE gels and transferred onto a nitrocellulose membrane (Amersham Pharmacia Biotech, Piscataway, NJ). The membranes were incubated overnight in blocking buffer at 4 °C with each antibody. Antibodies against sterol regulatory element-binding protein (SREBP)-1 and liver X receptor (LXR) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against SCD-1 and Elovl-6 were purchased from Abcam (Cambridge, MA). Antibodies against β-actin were obtained from Sigma (St. Louis, MO). The membranes were subsequently incubated with horseradish peroxidase-conjugated secondary antibody for 60 min at room temperature. Immunostaining with antibodies was performed using the Super Signal West Pico Chemiluminescent Substrate (Thermo Scientific, Hudson, NH) and detected with the LAS-3000 Plus (Fuji Photo Film, Tokyo, Japan). The samples were quantified and normalized against the β-actin control using ImageJ version 1.48.

Cells were lysed with RIPA buffer (50 mM Tris-HCl (pH8.0), 150 mM sodium chloride, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1% NP-40) containing cOmplete protease inhibitor cocktail (Roche). For standard immunoblotting analysis, aliquots of lysates were separated on 12% Tris-glycine gels as previously described [25 (link), 26 (link)]. For the detection of sAPPα and sAPPβ, aliquots of conditioned media were separated by 10% Tris-glycine gels, and probed with 6E10 and anti-sAPPβwt antibody, respectively. For the detection of APP-CTFs and Aβ, high-resolution electrophoresis was performed using 16% Tris-Tricine gels. APP-CTFs were detected with APPC15. The β1CTF and Aβ were detected by 82E1. The separated proteins on a membrane, were incubated with appropriate primary and secondary antibodies, and detected with ImmunoStar LD (Wako Pure Chemical Industries). For quantification chemiluminescence light signals were captured by a cooled charge-coupled device camera system (LAS-3000plus; Fuji Film).

Cells were washed twice with ice-cold PBS, resuspended in ice-cold RIPA buffer and incubated at 4 °C for 30 min. Lysates were centrifuged at 13,000 rpm for 20 min at 4 °C. Equal amounts of proteins were subjected to 7.5–15% sodium dodecyl sulfate polyacrylamide gel electrophoresis. The proteins were transferred to a nitrocellulose membrane (Amersham Pharmacia Biotech, Piscataway, NJ, USA) and reacted with each antibody. Immunostaining with antibodies was performed using the Super Signal West Pico (Thermo Scientific, Hudson, NH, USA) enhanced chemiluminescence substrate and detected with LAS-3000 Plus (Fuji Photo Film, Tokyo, Japan). Relative band intensities were quantified using ImageJ software version 1.53s (National Institute of Health, Bethesda, MD, USA).