Cell culture media were from GIBCO BRL. TOPRO-3 and phalloidin-Alexa 568 (Molecular Probes, Inc.) were used to label nuclei and actin. MDC was from Sigma-Aldrich. γ-Secretase inhibitors were from Calbiochem (X or L685,458), Elan (DAPT), and AstraZeneca (Compound C).
Polyclonal anti-PS1-NTF (B19.2), -CTF (B32.1) and -TLN (B36.1) have been described previously (Annaert et al., 2001 (link)). B63.1 and B59.1 were generated using a synthetic peptide mimicking the final 16 and 18 amino acids of APP and nicastrin, respectively, coupled to KLH (Pierce Chemical Co.). Mab 9C3 against nicastrin was produced by immunizing the same peptide in balb/c mice followed by generation of a hybridoma cell line according to established procedures. We acknowledge the antibody gifts of anti-calnexin (A. Helenius, ETH Zurich, Zurich, Switzerland), anti-ergic-53 (J. Saraste, University of Bergen, Bergen, Norway) -LC3 (T. Yoshimori, National Institute of Genetics, Shizuoka-ken, Japan), -Apg12 (N. Mizushima, National Institute for Basic Biology, Okazaki, Japan), PIP2 (G. Hammond, Cancer Research Institute, London, UK), -LBPA (J. Gruenberg, University of Geneva, Geneva, Switzerland), and -APP COOH terminus (c 1/6.1; P. Mathews, Nathan Kline Institute, Orangeburg, NY). Mabs to Lamp-2 (Abl-93) were obtained from Developmental Studies Hybridoma Bank (Iowa City, Iowa); anti-synaptophysin (cl.7.2) and anti-PS1-CTF (mAb 5.2) were from R. Jahn (MPI-Göttingen, Göttingen, Germany) and B. Cordell (Scios Inc., Sunnyvale, CA). mAbs to GM130 and EEA1 were from BD Biosciences, the transferrin receptor from Zymed Laboratories, β-COP from Sigma-Aldrich, and BIP from StressGen Biotechnologies.
Polyclonal anti-PS1-NTF (B19.2), -CTF (B32.1) and -TLN (B36.1) have been described previously (Annaert et al., 2001 (link)). B63.1 and B59.1 were generated using a synthetic peptide mimicking the final 16 and 18 amino acids of APP and nicastrin, respectively, coupled to KLH (Pierce Chemical Co.). Mab 9C3 against nicastrin was produced by immunizing the same peptide in balb/c mice followed by generation of a hybridoma cell line according to established procedures. We acknowledge the antibody gifts of anti-calnexin (A. Helenius, ETH Zurich, Zurich, Switzerland), anti-ergic-53 (J. Saraste, University of Bergen, Bergen, Norway) -LC3 (T. Yoshimori, National Institute of Genetics, Shizuoka-ken, Japan), -Apg12 (N. Mizushima, National Institute for Basic Biology, Okazaki, Japan), PIP2 (G. Hammond, Cancer Research Institute, London, UK), -LBPA (J. Gruenberg, University of Geneva, Geneva, Switzerland), and -APP COOH terminus (c 1/6.1; P. Mathews, Nathan Kline Institute, Orangeburg, NY). Mabs to Lamp-2 (Abl-93) were obtained from Developmental Studies Hybridoma Bank (Iowa City, Iowa); anti-synaptophysin (cl.7.2) and anti-PS1-CTF (mAb 5.2) were from R. Jahn (MPI-Göttingen, Göttingen, Germany) and B. Cordell (Scios Inc., Sunnyvale, CA). mAbs to GM130 and EEA1 were from BD Biosciences, the transferrin receptor from Zymed Laboratories, β-COP from Sigma-Aldrich, and BIP from StressGen Biotechnologies.
