The two-hybrid screening was conducted using the Matchmaker system from Clontech according to the manufacturer’s instruction. Yeast strain Y190 was transformed with the corresponding bait plasmids by the lithium acetate/polyethylene glycol 4000 procedure and selected on synthetic drop-out plates lacking tryptophan.
Two GAL4BD-APP baits were constructed using the pAS2 vector (Clontech). Construct pAS2-AT and pAS2-LK consisted of the COOH-terminal 58 amino acids or 48 amino acids of APP fused to the DNA binding domain (BD) of GAL4, respectively.
From the cDNAs isolated from the yeast two-hybrid screening, hJIP1 clone AT27 was cloned into Flag tagged pcDNA3.1 (Invitrogen), pECFP-N1 and pEYFP-N1 (Clontech) for expression in mammalian cells or in vitro.
Human JIP1e was constructed with the same sequence for hJIP1 (Genebank accession number AAD20443) with the first 24 amino acids exchanged for an alternative stretch of 34 amino acids derived from EST IMAGE:2545752 (ATCC). This was cloned into Flag tagged pcDNA3.1, pECFP-N1 and C1 and pEYFP-N1 and C1.
Fragments of hJIP1 were cloned by PCR using Pwo polymerase (Roche) and the following oligonucleotides (Life Technologies): SH3-forward: AAA AGA ATT CTG TTC TCC TGC ATC ATC; SH3-
reverse: AAA CTC GAG TTA GTC ACT GTT TTT GGC; PTB-forward: AAA AGA ATT CAG TTC CGG GTG AAG TTC CTG; PTB-reverse: AAA CTC GAG TTA CTC CAC AAA CTG CTT GTA.
HJIP1-SH3 containing residues 479-562 was cloned using primers SH3-forward and SH3-reverse, hJIP1 PTB containing residues 566-700 was cloned using primers PTB- forward and PTB- reverse and hJIP1-SH3PTB containing residues 479-700 was cloned using primers SH3- forward and PTB- reverse.
Appropriate restriction enzymes were used to splice these fragments into Flag tagged pcDNA3.1, pECFP-C1 and pEYFP-C1.
Mouse JIP1a in pCMV5 was obtained as a kind gift from Dr. Roger Davis (Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, Massachusetts)
Mouse JIP1b was constructed by cutting EST IMAGE:4506969 (Incyte Genomics) which codes for the 47 amino acid insertion with EcoO65I and EcoRV and splicing it into mJIP1a already cut with the same enzymes.
Human embryonic kidney (HEK) 293T cells were grown in RPMI 1640 media (Life Technologies) supplemented with glutamine and with 10% heat-inactivated fetal calf serum (Biofluids; Rockville MD).
Phosphorylation of fusion proteins was confirmed by western blotting with the PY20 monoclonal anti-phoshotyrosine antibody (Transduction Labs).
Transfected cells were immunoprecipitated for two hours at room temperature with either monoclonal antibody directed against the Flag epitope already bound to agarose beads (Sigma) or with 2.5 ml polyclonal Living-Colors antibody (Clontech) with 15ml protein A/G agarose beads (Pierce)
HEK 293T cells were plated in 24 well plates and co-transfected with the CFP and YFP fusion proteins using Fugene 6 (Roche).
All transfections with AID or AID(Y682G) had a DNA ratio of AID to JIP1 of 1:1 while all transfections with APP or APP(Y682G) had a DNA ratio of APP to JIP1 of 4:1.
A MoFlo Multi Laser Sorter (MLS), (Cytomation, Fort Collins, Co) was configured as follows. An argon laser tuned to 488nm was used to excite YFP (but doesn’t excite CFP) and a krypton laser tuned to 413nm was used to excite CFP.
YFP emission was detected with a 530nm/40nm bandpass filter and CFP emission was detected with a 473nm/12nm bandpass filter.
Adult BALB/c mice (age 3 months) were euthanized, brains were removed and homogenized in lysis buffer, protein was used for immunoprecipitations with aAPP (C7 or 1736, both kindly provided by Dr. D.J. Selkoe [Center for Neurologic Diseases, Harvard Medical School, Brigham and Women's Hospital])
JIP1-NT or rabbit anti-mouse IgG antibodies (ICN, Aurora, OH).