The following primary antibodies were used: rabbit anti-HB-EGF (antibodies-online), mouse anti-Myc (9B11; Cell Signaling Technologies), rabbit anti-EGF (antibodies-online), rabbit antipro-EGF (against aa 700-800; Novus Biologicals, R&D Systems Europe Ltd, England), rabbit anti-APP C-Terminal (clone CT695; Invitrogen), mouse anti-APP N-terminal (clone 22C11; Chemicon), mouse anti-APP residues 1-16 of Abeta (clone 6E10), rabbit anti-ERK1/2 (Millipore, Merck Life Science S.L.U., Portugal), rabbit anti-phosphorylated ERK1/2 (Thr202/Tyr204 Erk1 and Thr185/Tyr187 Erk2; Millipore), anti-acetylated alpha-tubulin (Sigma-Aldrich, Química S.A., Portugal), mouse anti-βIII Tubulin (Sigma-Aldrich). HRPconjugated anti-mouse and anti-rabbit secondary antibodies were from GE-Healthcare.
Secondary antibodies Alexa Fluor 405 goat anti-rabbit, Alexa Fluor 594 goat anti-rabbit or anti-mouse, and Alexa Fluor 488 goat anti-mouse were from Life Technologies. Human recombinant EGF was from Cell Signaling and was used at a 100 ng/mL working concentration (WC). The EGFR inhibitor drug PD 168393 (Sigma-Aldrich) was used at 10 µM WC. Texas Red-conjugated Transferrin was from Molecular Probes.
Secondary antibodies Alexa Fluor 405 goat anti-rabbit, Alexa Fluor 594 goat anti-rabbit or anti-mouse, and Alexa Fluor 488 goat anti-mouse were from Life Technologies. Human recombinant EGF was from Cell Signaling and was used at a 100 ng/mL working concentration (WC). The EGFR inhibitor drug PD 168393 (Sigma-Aldrich) was used at 10 µM WC. Texas Red-conjugated Transferrin was from Molecular Probes.