Anti adam10

Cells were lysed in LDS sample buffer (Invitrogen, Germany) including 100 mM dithiothreitol (Roth, Germany) and protease inhibitor mix (Roche, Germany). 20 µg proteins of whole cell lysate were separated on 10% SDS-acrylamide gels and transferred to a nitrocellulose membrane. Blots were either blocked with 5% BSA or milk powder and incubated with primary antibodies diluted in respective blocking buffer as follows: anti-APP (previously described: [35] (link)), anti-ADAM10 (Merck, Germany), anti-GSK3-beta (Bioss, Germany), anti-Pgp (Santa Cruz, Germany) anti-actin (Sigma, Germany), anti-P-ERK and anti-GAPDH (both: Cell Signaling, USA). Detection of APPs-alpha was performed as a dot blot with direct application of cell culture supernatant to the nitrocellulose membrane and 6E10 (Covance, Germany) as primary antibody. Blots were incubated with respective HRP-labeled secondary anti-mouse or anti-rabbit antibodies (Thermo Scientific, Germany) and GAPDH or actin were used as loading controls. Signals were detected with a CCD-camera imaging system and quantitatively analyzed with AIDA image analyzer 4.26 software (Raytest, Germany).