Phorbol-stimulated sAPPα secretion is not sensitive to the phospho-state of NSF at tyrosine 83. HEK293 cells stably expressing APP695 (HEK293–695) were transiently transfected with either wild type NSF (WT), the tyrosine 83 phospho state mimetic NSF mutant (Y83E), or the the tyrosine 83 dephospho state mimetic NSF mutant (Y83F). Empty vector was used as control (Cont). Cells were treated with PDBu for one hour after which media were collected and immunoprecipitated with antibody 6E10. Western blotting for sAPPα was performed using 6E10. (A) Representative Western blot for sAPPα in the absence (-) or presence (+) of PDBu. (B) Quantification of 3 such experiments.