Anti mouse p0260

Cells were lysed in lysis buffer (150 mM NaCl, 50 mM Tris/HCl pH 7.4, 2 mM EDTA, 0.1% NP-40, 0.1% Triton-X 100) containing protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany), adjusted to equal protein amount and loaded on 10–20% Tricine gels (Anamed Elektrophorese, Groß-Bieberau, Germany). Proteins were transferred onto nitrocellulose membranes (Whatman, Dassel, Germany). For Western blot analysis the following primary antibodies were used: anti-sAPPβ MBS492139 (MyBioSource, SanDiego, USA), anti-NEP ab951 (Abcam, Cambridge, UK) and W02-antibody (Millipore, Billerica, USA) for detection of sAPPα and immunoprecipitated Aβ as described earlier (Grimm et al., 2011b (link), 2015 (link)). Anti-rabbit W401 (Promega, Mannheim, Germany) and anti-mouse P0260 (Dako, Hamburg, Germany) were used as secondary antibodies. Proteins were detected by ECL-method (Perkin Elmer, Rodgau-Jügesheim, Germany), densitometric quantification was performed with Image Gauge V3.45 software.

Samples used for the WB experiments were adjusted to equal protein concentration in advance.
For the determination of BACE1, cell lysates were prepared by lysing cells in 150 mM NaCl, 50 mM Tris/HCl pH 7.4, 2 mM EDTA, 0.1% NP-40, 0.1% Triton-X 100. For the determination of total secreted Aβ level and sAPPβ conditioned media were used.
The following antibodies were used for WB analysis: W02 antibody (5 μg/mL; Millipore, Billerica, MA, USA), anti-sAPPβ: Mbs492139 (1:250; MyBioSource, San Diego, CA, USA), BACE1: ab2077 (1:1000; abcam, Cambridge, UK), anti-actin ab1801 (1:1000; abcam), anti-rabbit IgG HRP Conjugate W401B (1:5000; Promega, Mannheim, Germany) and anti-mouse P0260 (Dako, Hamburg, Germany).
Aβ levels were detected by performing immunoprecipitation of conditioned media before WB analysis. Therefore, 20 μL protein G-Sepharose and W02 antibody (5 μg/mL) were used.
Enhanced chemiluminescense (ECL)-method (Perkin Elmer, Rodgau-Jügesheim, Germany) was used to detect proteins. Densitometrically quantification was performed by using Image Gauge V3.45 software (Fujifilm, Düsseldorf, Germany).

BACE1 and PS1 protein levels were analyzed in cell lysates, whereas for the determination of the total protein levels of the secreted proteins Aβ, sAPPα, and sAPPβ growth media were used. All samples were adjusted to the same protein amount via BCA. Antibodies and dilutions used in this study are: W02 antibody for the detection of β-CTF and total Aβ (5 µg/mL; Millipore, Billerica, MA, USA), anti-human sAPPβ (1:50; IBL America, Minneapolis, MN, USA), anti-human BACE1 B0806 (1:1000; Merck, former Sigma-Aldrich, Darmstadt, Germany), anti-human PS1 sc-7860 (Santa Cruz, Dallas, Texas, US), anti-rabbit IgG HRP Conjugate W4011 (1:5000; Promega, Mannheim, Germany) and anti-mouse P0260 (Dako, Hamburg, Germany). For detection of proteins, the enhanced chemiluminescense (ECL)-method (Perkin Elmer, Rodgau-Jügesheim, Germany) was used. The quantification was performed densitometrically with Image Gauge V3.45 software (Fujifilm, Düsseldorf, Germany).