Minopoli G, de Candia P, Bonetti A, Faraonio R, Zambrano N, Russo T. The beta-amyloid precursor protein functions as a cytosolic anchoring site that prevents Fe65 nuclear translocation. J Biol Chem. 2001 Mar 2;276(9):6545-50. PubMed Abstract, View on AlzSWAN

REAGENTS/MATERIAL:
The GFP-Fe65 and Fe65-HA fusion proteins encoding wt Fe65 obtained by amplification of the rat Fe65 cDNA as described in Faraonio et al. The Fe65 deletion mutants GFP-Fe65D666-711, GFP-Fe65D538-711, GFP-Fe65D1-287, GFP-Fe65D1-253, as well as the fragment corresponding the Fe65 cDNA codons 191-290, were obtained by amplication of the Fe65 cDNA with specific oligonucleotide primers (CEINGE). The Fe65 point mutant C655F cDNA was obtained by using QuickChange Kit (Stratagene). The CMV-Fe65-HA vector expressing wt rat Fe65 tagged with the HA epitope was obtained by cloning in the HindIII site of pRC-CMV (invitrogen). COS-7 cells were cultured in Delbecco's modified Eagle's medium (Life Technologies, Inc.) PC12 cells were cultured in RPMI (Life Technologies, Inc.) supplemented with 10% horse serum, 5% fetal bovine serum, and 1% penicillin/streptomycin mixture. APP was stained with 6E10 or 369 antibodies, using Texas red-conjungated secondary antibodies (Jackson ImmunoResearch Laboratories). Fe65-HA was stained with the anti-HA polyclonal antibody Y-11 (Santa Cruz Biotechnology). For immunoprecipitation, cell extracts were incubated with monoclonal antibody anti-GFP JL8 (Clontech).

FUTURE DIRECTION:
Further experiments are needed to evaluate whether the APP processing by g-secretase could result in the targeting of Fe65 to the nucleus through the cleavage of the transmembrane domain of APP.