Methods

cDNA cloning
cDNA cloning of the Acdp gene family was performed based on human homologue sequences as previously reported [2]. Briefly, the human ACDP cDNAs and predicted AA sequences were used to search the mouse EST database for EST markers corresponding to each Acdp member. A forward primer within the human ACDP 5' cDNA coding region (after start codon) and a mouse reverse primer from the mouse EST marker were used to amplify the homologue sequence from mouse cDNA at very low annealing temperature (45–50°C). A nested PCR using an inside reverse primer from the mouse EST sequence and the same human forward primer was then carried out to amplify the specific mouse gene from the first round PCR products at high annealing temperature (62°C). The expected PCR products were directly excised from agarose gel and sequenced by an ABI377 automatic sequencer. The sequence was further confirmed using a forward primer from newly identified sequence and a reverse primer from known sequence. Once most of the coding sequences were identified, partial sequence of exon 1 and the 5' UTR sequences were obtained by BAC DNA sequencing.

Northern blot analyses
Multiple Choice Northern Blot filters containing 12 different mouse tissues were purchased from Origene. The filters were probed for each Acdp gene with a PCR fragment (around 350 bp) from last exon and the 3' untranslated region labeled with α-32P dCTP using the random primer extension system (Life Technologies). Hybridizations were carried out overnight. The filters were washed twice with washing buffer I (2×SSC, 0.1% SDS) at 42°C for 15-min, and then washed twice with washing buffer II (0.25×SSC, 0.1% SDS) at 65°C for 15-min. Washed filters were exposed to X-ray films for overnight or longer [3,4].

Antibody production and Western blot analyses
Peptides linked to KLH (keyhole limpet hemacyanin) from Acdp1 N- and C-terminals and the conserved domain (ACD) were used for generation of antibodies specifically for Acdp1 and all Acdp members as reported, respectively [5]. Western blots were carried out Using ECL (PIERCE) as described previously [1]. The membranes were washed extensively after incubation with primary and secondary antibodies and were then developed with X-ray films with optimal exposure time.

Chromosome localization
The T31 mouse radiation hybrid panel from Research Genetics was used to map the chromosome location of each Acdp member. Primers from 3' UTR of each Acdp member were used to amplify the 100 radiation hybrid clones representing the mouse genome. The data were submitted to the Jackson Laboratory Mouse Radiation Hybrid Database for analysis.

Sequence analyses
Sequence assembly was performed with program Sequencher (Gene Codes Corp). Protein and DNA homology searches were carried out with tblastn, tblastx, blastp and blastn programs . Multiple sequence alignments were performed with GeneDoc and pairwise sequence alignment . Multiple programs including BCM Search Launcher , ProfileScan , sequence motif search , ExPASy  and 3Dpssm  were used for searching sequence features of known protein. Phylogenetic tree was constructed by Clustalw program (version 1.81) using UPGMA (Unweighted Pair Group Method using Arithmetic averages) algorithm [14].

Neuronal cell preparation and immunostanining
Hippocampal neuron cultures were prepared as previously reported [6]. In brief, the hippocampuses were dissected out from mouse embryos at 16 days in utero. The tissues were then incubated for 20 min at 37°C in MEM (minimum essential medium) modified for suspension culture (Life Technologies) plus 0.25% trypsin (Life Technologies). The dissociated hippocampal neurons were plated on glass coverslips coated with a confluent monolayer of mouse cortical astrocytes obtained as described below. The neurons were maintained at 37°C in a humidified atmosphere with 5% CO2. Cortical astrocytes dissociated from newborn mouse cortices were grown in culture flasks at 37°C in a humidified atmosphere with 5% CO2 until confluent. The cells were exposed to 10-5 M cytosine arabinoside (Sigma) and cultured for additional 12–24 hrs at 37°C. After remove of the media with cellular debris, the cells were used for coating coverslips.
For immunostaining, neuronal cells on the coverslips were first fixed in PBS containing 4% paraformaldehyde (PFA) for 12 hrs at 4°C and then incubated in a solution containing 4% PFA and 0.4% Triton X-100 at 4°C for 1 hr. After washing with PBS three times, the cells were incubated with a blocking solution containing 1:30 normal goat serum, and subsequently incubated with a rabbit polyclonal anti-ACDP antibody (1:3000) overnight at 4°C. After extensive washing with 1% goat serum PBS solution, the cells were incubated with an Alex 488 conjugated secondary antibody (1:100 in 1% goat serum PBS solution, Molecular Probes) for 3 hrs at room temperature. Following final washes with 1% goat serum PBS solution, the neuronal cells on the coverslips were cover-slipped with a glycerol-based anti-photobleach medium. The cells were viewed under a confocal microscope (Carl Zeiss). Images were captured with a CCD camera and acquired by the Scion Image software (Scion Corporation, Frederick, MD).

Gene bank accession number
The cDNA sequences for the Acdp gene family have already been deposited in gene bank with accession numbers AF202994 (Acdp1), AF216961 (Acdp2), AF216964 (Acdp3) and AF216963 (Acdp4).