Whirlin (Whrn) is a PDZ-containing protein expressed throughout the central and peripheral nervous system. A. Schematic showing the relative organization of the twelve exons which make up the Whrn full-length sequence including untranslated exon regions (white boxes) and coding sequence (alternating grey boxes). Whrn’s second, short isoform begins with an alternative transcriptional start site (asterisks). Both variants contain PDZ-domains (yellow boxes) and a Proline-rich domain (blue box). A red rectangle highlights the region used for antibody creation (RbWhrn349). B. RT-PCR analysis shows absence of any Whrn transcripts in homozygous Whrn exon 1 knockout mice from dorsal root ganglia (DRG, peripheral neuronal nuclei), sciatic nerves (SN, peripheral glial nuclei), and spinal cords (SC, combination neuronal/glial nuclei). mRNA transcripts were reverse transcribed and amplified using primers located on Whrn exon 1 and 4 (top panel), Whrn exon 9 and 10 (middle panel), or actin (bottom panel). C. Relative quantification of Whrn mRNA from Figure 1B expressed as a ratio of Whrn to actin band intensity. D. Immunoblots of 110 kDa Whrn protein band in wild-type and Whrn mutant DRG, SN, SC, and whole eye using affinity-purified RbWhrn349 antibody. αTubulin served as a loading control. E. Immunoblots of wild-type and Whrn knockout mutant 4, 6, 8, and 16-week-old spinal cord lysates. The expression profile using various myelinated axonal domain markers includes Caspr, Neurofascin (186 and 155), 4.1B, Caspr2, as well as CASK. αTubulin served as a loading control. F. Immunofluorescence of teased sciatic nerve fibers from wild-type (upper panel) and Whrn knockout mice (lower panel) mice. Neurofascin (NFCt, red) and paranodal Caspr (green) reveal paranodal compaction is disrupted in Whrn knockout fibers. Note NFCt (red) detects both paranodal NF155 and nodal NF186 isoforms.
Green et al. BMC Neuroscience 2013 14:96 doi:10.1186/1471-2202-14-96