sodium deoxycholate; 0.1% SDS; 50 mM Tris, pH8) and NP40 (150 mM NaCl; 1% NP-40; 50 mM Tris, pH8) lysis buffers supplemented with a protease inhibitor cocktail (Roche), and subsequently quantified using the Qubit Fluorometer (Invitrogen). Hemagglutinin epitope signal was easier to detect in NP40 lysates, while RIPA provided clearer results for the NAIP-specific antibody. Bi-phased gels containing TEMED and APS (4% stacking, 9% separating) were used to resolve total cellular protein in electrophoresis running buffer (106: 25 mM Tris; 192 mM glycine; 0.1% SDS). Subsequently, separated proteins were transferred using a Hoefer TE 22 tank transfer unit (Amersham Biosciences) onto Immobilon-P PVDF membrane (Millipore) in fresh transfer buffer (25 mM Tris, 192 mM glycine, 10% methanol, 0.1% SDS). To assess NAIP protein isoforms in primary human tissues an IMB-103-50 Instablot membrane was purchased from Imgenex (San Diego). Blocking of all membranes was performed in 5% reconstituted skim milk powder under constant agitation at 4u overnight. The following morning, blocking solution was replaced and fresh primary antibodies were applied at 1:1000 NAIP (Abcam), 1:3500 Actin (Sigma), and 1:3500 HA (BAbCO) for one hour at room temperature under constant agitation. Washes were carried out with TBS-T (106: 20 mM Tris;1.4 M NaCl;1% Tween-20) at room temperature in 5 minute intervals, no more than five times. Secondary antibody was diluted in fresh TBST and 1% blocking solution to a final concentration of 1:100 000, and incubated for one hour at room temperature under constant agitation. Washes were conducted as above. Proteins were detected using the Enhanced Chemilumi- nescence Kit (Perkin Elmer) and Kodak BioMax Film and cassettes (Kodak). Where necessary the Instablot was stripped with 0.2 M NaOH, all other membranes were cleared by an acidic strip solution (25 mM glycine-HCl pH2, 1% SDS). Computational tools Dot plots. Analysis of the underlying DNA sequence of 5q13.3 was performed to better understand the exons mapping to particular NAIP copies. DNA sequences were obtained from the UCSC Human Genome Browser March 2006 (hg18) assembly [34]. The genomic sequence of NAIPfull (chr5:70,298,269-70,360,000) was used to assess exon architecture of the remaining copies: NAIP1 (chr5:70,425,120- 70,469,539); NAIP2 (chr5:69,424,009-69,495,811); and yNAIP1 and 2 (chr5:69,780,634-69,828,298; 68,921,612-68,967,595). Indicated sequences were compared using the web-based jdotter (http:// athena.bioc.uvic.ca/workebnch.php?tool+jdotter&db = ). Sequence Analysis. Sequenced clones were uploaded, managed, and analyzed in the SDSC Biology Workbench (http://workbench.sdsc.edu). Precise mapping of the clones to the human genome was completed using the BLAT tool in the UCSC Genome Browser [34]. ORF prediction. Sequences of interest were scanned for open reading frames using NCBI’s ORF Finder, and subsequent analysis of encoded domains was completed with BLASTP. Supporting Information Figure S1 Homology of human NAIP copies. Dot plots were performed to better understand the exon architecture of each NAIP copy. The NAIPfull copy in the 2006 assembly of the human genome (70,298,269–70,360,000) was compared to the genomic sequence underlying the other NAIP copies (as indicated). The coordinates of tested sequences are shown. Found at: doi:10.1371/journal.pone.0005761.s001 (4.44 MB TIF) Figure S2 Unequal levels of NAIP 59 and 39 transcription. Semi- quantitative RT-PCR was performed at a low cycle number across a panel of human tissues to determine the levels of NAIP 59 and 39 Novel Promoters for Human NAIP transcription. Red arrowheads indicate localization of the primers used in this experiment, and are shown relative to a diagram of NAIPfull, at bottom. Found at: doi:10.1371/journal.pone.0005761.s002 (7.43 MB TIF) Figure S3 Analysis of NAIPfull transcription. A) NAIPfull- associated TSS are shown (bent arrows) as previously described: i and ii [15]; and iii [36]. Black boxes indicate exons, and labeled boxes represent LTRs (shaded) and SINEs (speckled). Colored arrowheads indicate tiled primers used to better understand the TSS associated with NAIPfull transcription in THP1 cells [36]. B) Tiled-primer experiments in the indicated primary human tissues and cell lines. The primers used are color-coded with those shown above (A). Primary tissues were Southern blotted to increase resolution, using a radio-labeled oligonucleotide specific for a region of exon 1 common to all isoforms. Found at: doi:10.1371/journal.pone.0005761.s003 (10.09 MB TIF) Figure S4 Sequence analysis underlying NAIP transcription start sites for the novel NAIPSg (A), NAIPJb (B), and NAIPGUSBP1 (C) regulatory regions. cDNA sequence is shown in capitalized letters and the underlying genomic DNA (gDNA) is shown in lower case. Subscript numbers associated with green (Alu) or purple (L1) font in the gDNA track denote positions along the relevant transposable element. All discovered transcription start sites are indicated in black bold-face, and superscript numbers in B and C represent the number of clones arising from the particular position. Vertical dashed lines in A, B, and C represent exon junctions, and slight extension of gDNA underlying exon junctions indicates the appropriate splice donor and acceptor sites. Splicing of NAIPJb clones does not occur and transcription proceeds through intervening intron 9 into exon10. Red bold-faced letters in A and B indicate sites of RNA-editing. Potential regulatory motifs are shown relative to the lower case genomic DNA sequences as follow: TATA box - italics; Initiator sequences - overlines; Downstream promoter elements - underlines [39]; yellow, light blue, and dark blue shading denote estrogen response element, retinoic acid response element, and AP-1 binding motifs, respectively [13]. Found at: doi:10.1371/journal.pone.0005761.s004 (0.05 MB DOC) Figure S5 Broad transcription of novel NAIP isoforms. RT-PCR was performed to determine the breadth of expression of NAIP from the Alu and GUSBP1 39 UTR-contained TSS, represented by bent arrows. Color-coded arrows indicate the primers used: expression from NAIPSg is indicated by blue arrows and box; expression from NAIPGUSBP1 is indicated by purple arrows and box; and expression from NAIPJb is indicated by orange arrows and box. No splicing is observed between the AluJb transcription start site and the adjacent downstream exon; +/2 RT controls indicate low, or no, contamination of genomic DNA. Diagrams are not drawn to scale. Found at: doi:10.1371/journal.pone.0005761.s005 (7.82 MB TIF) Figure S6 NAIP protein sequence and encoded domains. The protein sequence of NAIPfull is shown, and exon boundaries are indicated by numbers above circled arrows. Potential downstream in-frame initiation codons are indicated in red font, and the surrounding nucleotide sequence is shown beneath, with ‘atg’ in boldface. Underlines represent start codons with a sequence context in general agreement with derived consensi [42,43]. The stop codon is denoted by an asterisk. Yellow, purple, and green highlighting indicates BIR, NBD, and LRR domains, respectively. Found at: doi:10.1371/journal.pone.0005761.s006 (0.03 MB DOC) PLoS ONE | www.plosone.org 10 June 2009 | Volume 4 | Issue 6 | e5761