The clathrin mediated endocytic pathway is not involved in oligomeric Aβ42-induced neurotoxicity. A. N2A cells were transiently transfected with siRNA for clathrin heavy chain, treated with 10 μM oAβ42 for 24 hours and assayed for neurotoxicity as detected with an ATP-based luminescence cell viability assay (CellTiter-Glo, Promega); no difference with treatment. Inset, clathrin heavy chain levels were characterized with clathrin heavy chain antibody (Sigma) by Western blot analysis with an equal amount of lysates from cells transfected with non-targeted siRNA and CLTC siRNA. B. N2A cells were transiently transfected with wild type AP180-FL or dominant-negative AP180-CT construct, treated with 10 μM oAβ42 for 24 hours, and assayed for neurotoxicity; no difference with treatment. Inset, AP180-CT was detected with Flag antibody (Sigma) by Western blot analysis with an equal amount of lysate from cells transfected with AP180-FL and AP180-CT. C. N2A cells were transiently transfected with AP180-FL or AP180-CT. 48 hours post-transfection, cells were treated with 10 μM oAβ42 for 30 minutes, fixed and stained for Aβ with Aβ42 specific antibody (Invitrogen, green). AP180-FL and AP180-CT mutant transfected cells were identified by anti-Flag antibody (red). Nuclei appear blue as detected by DAPI staining. Cells were individually outlined and mean fluorescence intensity of Aβ signals was quantified with NIH image software. There were similar levels of Aβ accumulation in these transfected cells.