were probed with affinity-purified rabbit anti-ELMOD1 antisera (1:500) followed by anti-rabbit IgG, horseradish peroxidase (HRP)-linked F(ab9)2 fragments from donkey (1:50,000; GE Lifesciences, Piscataway, NJ). ECL Plus western blotting detection reagents (GE Lifesciences) were used for enhanced chemilumines- cent detection of specifically bound antibody by exposure to audioradiograph film (Eastman Kodak Co., Rochester, NY). DIG-labeled riboprobes and mRNA in situ hybridization DNA sequences of primers for amplifying a 768 bp fragment of Elmod1 cDNA from brain tissue were obtained from the Eurexpress Transcriptome Atlas Database for Mouse Embryo (www.eurexpress.org, Template ID T39634) [71]. The sequence for the SP6 promoter was added as a 59overhang to the reverse primer so that the antisense probe could be generated directly from the PCR product. A digoxigenin-11-UTP (DIG) labeled antisense riboprobe was produced from the purified PCR product by in vitro transcription according to the manufacturer’s instructions (Roche Applied Science, Indianapolis, IN). In situ hybridization was performed as described [72] with a the following modifications. Tissue was fixed overnight at 4uC in 4% paraformaldehyde. Sections were mounted on Superfrost plus slides (Fisher Scientific, Suwanee, GA). Slides were air dried at room temperature for 1–2 hours, followed by fixation in 4% parafomaldehyde for 10 minutes. Digestion was carried out for 10 min at room temperature in buffer consisting of 50 mM Tris, pH 7.5, 5 mM EDTA, and 1 mg/ml proteinase K followed by fixation in 4% paraformaldehyde for 5 min. Hybridization was performed at 65u C overnight in buffer consisting of 50% deionized formamide, 0.3 M NaCl, 20 mM Tris, pH 8.0, 5 mM EDTA, pH 8.0, 10% dextran sulfate, 16 Denhardt’s solution, 0.5 mg/ml tRNA from baker’s yeast (Sigma, St. Louis, MO), and 500 ng/ml of the denatured DIG labeled probe. Washes were also carried out at 65u C. Following immunological detection [72], slides were mounted with VECTASHIELD mounting medium (Vector Laboratories, Burlingame, CA). Supporting Information Figure S1 Analyses of Elmod1 alternative transcripts and promoter site. A. Exon structure of the Elmod1 gene and alternative splicing. White rectangles (proportionately sized) represent individual exons of the reference sequence NM- 177769 and are numbered according to their linear sequence on Chr 9. Intronic regions are not shown, but splicing events are represented as lines connecting the ends of exons. Exon regions from alternatively spliced transcripts are indicated by black rectangles. The mouse cDNA sequences AK029207, BC120566, BC144961, and BC049160 include an additional 38 bp at the 59 end of exon 3 (indicated by black rectangle labeled ‘‘a’’), presumably the result of an alternative splice acceptor site. Our analysis of PCR products from cDNA templates using exon 1 and exon 3 specific primers indicates that the shorter 146-bp exon 3 is the most predominant isoform in adult mouse brain tissue, although transcripts with the extended 184-bp exon 3 are detected in low abundance (lane 1 in panel B). Reference sequences for the human ELMOD1 gene are derived from two transcripts: a 2990 bp isoform 1 (NM_018712), which is transcribed from 12 exons and encodes a 334 aa protein, and a 2967 bp isoform 2 (NM_001130037), which is transcribed from 11 exons and encodes a 326 aa protein. The NM_018712 isoform includes an additional, in-frame 24-bp exon that lies between exons 8 and 9 of NM_001130037 (indicated by black rectangle labeled ‘‘b’’). None of the reported mouse cDNAs or ESTs includes this extra exon; Elmod1 Mutations Cause Inner Ear Dysfunction however, we found evidence for it by exon-specific PCR analysis of cDNAs from mouse brain tissue, although it is much less abundant than transcripts lacking this alternative, additional exon (lane 2 in panel B). The mouse AK029207 cDNA sequence for Elmod1 includes an alternative last exon (11A, indicated by black rectangle labeled ‘‘c’’ in A), which is different from the standard last exon (11S) of NM_177769 and most other cDNAs and spliced ESTs. We did not detect this alternative exon 11 in our PCR analyses of cDNA from mouse brain tissue using exon 10 primers and primers specific for the alternative exon 11 of AK029207 (Table S2). B. Alternative, low abundance Elmod1 transcripts detected in mouse brain tissue. Lane 1 shows PCR products from adult mouse brain cDNA obtained with primers wex1F and wex3R2. These primers are specific to exons 1 and 3. The PCR product size for the predominant transcript, which contains the smaller exon 3 (146 bp), is expected to be 325 bp, whereas the product size for the alternative transcript (marked by asterisk), which contains the additional 38 bp (designated 3A in panel B) of the extended exon 3, is expected to be 363 bp. Lane 2 shows PCR products from the same cDNA obtained with primers wex7F and wex10R. These primers are specific to exons 7 and 10. The PCR product for the predominant transcript is expected to be 236 bp; whereas the less abundant PCR product (marked by asterisk) is expected to be 260 bp because the alternative transcript includes an additional 24 bp exon between the standard exons 8 and 9. The lane marked ‘‘bp’’ is a 100 bp size ladder with 100 bp and 500 bp positions indicated by arrows. The DNA sequences of PCR primers are given in Table S2B. C. Transcription start site detected in mouse brain tissue. A 59 RACE assay with brain-derived cDNA and nested primers specific to exon 2 was used to determine the transcription start site of Elmod1. Lane 1 shows the 250 bp PCR product that was produced with the 59 RACE outer primer and a gene-specific outer primer (wex2R1), and Lane 2 shows the 220 bp product that was produced with the 59 RACE inner primer and a gene-specific inner primer (wex2R2). Lane 3 is a negative control with no cDNA, and Lane 4 is a positive control showing the 195 bp product expected with two gene-specific internal primers (wex1F and wex2R2). The DNA sequences of PCR primers are given in Table S2B. The lane marked ‘‘bp’’ is a 100 bp size ladder with 100 bp and 500 bp positions indicated by arrows. We identified a single transcription start site corresponding approximately to nucleotides 120–130 of NM_177769. Consistent with this result, we were unable to produce RT-PCR products with primers corresponding to cDNA sequences that are 59 of this region. (TIF) Figure S2 In situ expression of Elmod1 mRNA in brain and eye. Expression of Elmod1 was detected in specific regions of the brain in wildtype (+/+) mice, including Purkinje cells (PCs) of the cerebellum (A). Elmod1 expression also was detected in the hippocampus, where it was localized to the granule cell layer (Gcl) of the dentate gyrus and pyramidal neuron cell bodies (Pyr) of the cornu ammonis (B). Shown are brain sections of postnatal day 45 (P45) mice, but similar expression patterns were seen at P7 and P15. Elmod1 expression was also detected in retinal ganglion cells (RGCs) in the eyes of +/+ mice at P2 (C) and at P15 (not shown). Mutant rda/rda mice served as negative controls for probe specificity, as seen by the lack of detectable Elmod1 expression in the cerebellum of rda/rda mice (D). All panels are at the same magnification; the scale bar represents 250 micrometers. (TIF) Table S1 Genetic map position of the rda mutation on mouse Chr 9. PLoS ONE | www.plosone.org 11 April 2012 | Volume 7 | Issue 4 | e36074