Calbindin-D32k in the Echinoderm Nervous System Table 1. Antibodies used in this study. Antigen Calbindin 1 Parvalbumin Calbindin 2 GFSKLYamide Galanin b-tubulin Raised in rabbit (polyclonal) rabbit (polyclonal) rabbit (polyclonal) rabbit (polyclonal) rabbit (polyclonal) mouse (monoclonal) Tubulin from rat brain Unknown (RN1) mouse (monoclonal) Homogenate of the radial nerve of H. glaberrima For more detailed information, see Materials and Methods. doi:10.1371/journal.pone.0032689.t001 Immmunogen 28 kDa calbindin-D protein purified from rat kidney Abcam (ab11426) Parvalbumin from rat skeletal muscle Full length Calretinin GFSKLYa synthetic peptide Galanin Source Affinity Bioreagents (PA1-933) Abcam (ab702) Dr. Garc´ıa-Arrara´s lab [30] Dr. Garc´ıa-Arrara´s lab [31] Sigma (T-4026) Dr. Garc´ıa-Arrara´s lab [27] Dilution used 1:500 1 mg/ml 1:100 1:1,000 1:1,000 1:500 1:100,000 examined and photomicrographs taken on a Nikon Eclipse E600 fluorescent microscope with FITC, R/DII and DAPI filters. Images were recorded using the MetaVue software (version 6.0; MDS Analytical Technologies, Toronto, Canada) or the Spot Basic software (version 4.7; Diagnostic Instruments, Sterling Heights, MI), and Image J (version 1.37; NIH, Bethesda, MD). These were cropped, brightness and contrast adjusted, using Adobe Photoshop 7.0 (Adobe Systems, San Jose, CA). Bioinformatics Sequences were downloaded from the NCBI GeneBank protein database and stored in Biology Workbench v3.2 [32]. BlastP were performed against the public nonredundant protein database in GeneBank. EF-hand domain determinations were performed using ScanProsite [33]. Alignments were carried out with ClustalW and edited with GeneDoc CLC free-workbench software (v.3.2.1 by CLC bio A/S, Denmark), or by using the Blosum62 matrix and edited with Geneious software (v.5.4.6 by Biomatters LTD, New Zealand) [34]. The phylogenetic trees were construct- ed using the unweighted pair group method with arithmetic mean (UPGMA) with default parameters and distances calculated using the Jukes-Cantor genetic distance model using the Geneious software (v.5.4.6 by Biomatters LTD, New Zealand) [34]. Western blots The western blot assay protocol used has been described previously [27]. Briefly, approximately 300 mg of radial nerve homogenates were run at 100 V in SDS-PAGE (5% stacking and 15% running gel) for 2 h using a BioRad Mini Protean Electrophoresis system. The gel was equilibrated for 10 min in Towin buffer and transferred to a PVDF membrane at 100 V for 1 h in a Mini Trans Blot Cell (BioRad, Hercules, CA). The membrane was then blocked for 1 h at room temperature with 5% nonfat dry milk and washed three times in Tris-buffered saline with 0.1% Tween-20. The membrane was then incubated overnight with the respective primary antibody. The primary antibodies used were anti-calbindin 1 (1 mg/ml); anti-parvalbumin (1 mg/ml); anti- calbindin 2 (1:100), diluted with RPMI 1640 medium supple- mented with 5% horse serum. Positive controls in which mouse kidney homogenates were treated with the anti-calbindin 1 and the anti-calbindin 2 primary antibodies, and rat skeletal muscle homogenates with the anti-parvalbumin primary antibody were done to corroborate the quality of the homogenates and the antibody binding. Negative controls were done by using the EF- hand domain-containing synthetic protein EF-hand domain protein 2 (EFHD2) [35]. Following primary antibody incubation, the membrane was washed three times for 5 min each, and then incubated for 1 h in sheep anti-mouse (1:10,000 dilution) or donkey anti-rabbit (1:20,000 dilution) IgG peroxidase-linked secondary antibody (Amersham Biosciences, Piscataway, NJ) diluted with RPMI 1640 supplemented medium. After three washes of 5 min each, the membrane was incubated for 5 min in Super Signal West Dura Chemiluminescent Substrate (Thermo Fisher Scientific, Rockford, IL) and the signal was visualized using a Chemi System (UVP BioImaging Systems). Cloning of the H. glaberrima calbindin-D32k cDNA used in the cloning was prepared from H. glaberrima radial nerve extracts. After dissection of the radial nerves, the tissues were placed in 1 ml of TRIzol Reagent (Invitrogen, Calsbad, CA) and homogenized with a PowerGen Model 125 Homogenizer (Thermo Fisher Scientific, Rockford, IL). Following a phenol-chloroform extraction, total RNA was obtained from the aqueous supernatant. cDNA was then synthesized from the total RNA using M-MLV Reverse Transcriptase (Invitrogen, Calsbad, CA) and oligo(dT) primers. Degenerate primers (F59-GACCA- GAATAAGGATGGAAAG-39, R59-ACTCTGCCCCGTGAT- TTT-39) were designed against a potential calbindin 2 homolog found in the S. purpuratus genome (XP_001178717) and used to amplify a 376 bp fragment from H. glaberrima radial nerve cDNA. A highly conserved region was chosen for amplification by aligning sea urchin calbindin 2-like protein (XP_001178717) with calbin- din-D32k from D. melanogaster (NP_476838) and calbindin 2 from M. musculus (NP_033918). A start and stop codon were affixed to the 59 and 39 positions of the insert, respectively, using PCR amplification (F59-ATGGACCAGAATAAGGATGGAAAG-39, R59-TCAACTCTGCCCCGTGATTTT-39). Using this template, the restriction sites BamHI and NotI were affixed to the 59 and 39 positions, respectively, using PCR (F59-GAAATAGGATC- CATGGAC CAGAATAAGGATGGAAAG-39, R59-TTCCCC- GCGGCCGCTCAACTCTGCCC CGTGATTTT-39). This product was purified using a QIAGEN Spin Column (QIAGEN, Valencia, CA) and inserted into pGEX6P-1 (GE Healthcare Biosciences, Piscataway, NJ), using BamH1 and NotI restriction enzymes (New England Biolabs, Ipswich, MA). The vector was previously treated with Calf Intestinal Alkaline Phosphatase (Invitrogen, Carlsbad, CA) diluted to 0.01 u/uL in Buffer CIAP. The ligation was carried out using T4 ligase (New England Biolabs, Ipswich, MA), and the construct was heat shocked into OneShot BL21 Star (DE3) chemically competent E. coli cells (Invitrogen, Carlsbad, CA). After the shock, vials were incubated PLoS ONE | www.plosone.org 3 March 2012 | Volume 7 | Issue 3 | e32689