Effect of PKA inhibitors on the βAPP maturation by wtβAPP-expressing HEK293 cells. Stably transfected HEK293 cells overexpressing wild type βAPP were incubated for 7 h at 37°C in the absence (control) or in the presence of the indicated concentrations of H89 or myristoylated PKI. Secreted APPα (D) was immunoprecipitated with 207 antibody and then was analysed by SDS–PAGE, Western blotted and revealed with mAb2H3 as described in the Methods. Intracellular full-length βAPP (βAPPFL, A) and p10 (E) were identified by Western blot analysis with WO2 and BR188 antibodies, respectively. For Aβ detection, cells were preincubated with PKA inhibitors as above for 1 h then metabolically labelled for 6 h in the presence of the inhibitors. Aβ42 and x-42 (C) and Aβ40 and x-40 (B) were sequentially immunoprecipitated as described in the Methods, with FCA3542 and FCA3340, respectively. (F) Illustrates the quantitative densitometric analyses expressed as the per cent of control obtained in absence of inhibitors. Values are the means of four independent experiments.