Figure 1

Expression of LDB3 splice isoforms in brain and spinal cord. (A) Expression levels of Ldb3 mRNA in brain (blue circle) and spinal cord (red square) of 10-day (P10)- and 6 month-old mice measured with ddPCR assays using equal amounts of input cDNA per reaction. The bar graph with scatter plot represents mean ± S.D.; n = 3 mice; triplicate assay; a two-way ANOVA with the Bonferroni correction ****p < 0.0001. (B) A representative immunoblot of LDB3 isoforms in human motor cortex and spinal cord detected by a rabbit monoclonal antibody (Abcam; ab171936). Multiple bands are seen between 37 and 75 kDa (arrows). Two-fold higher amount of proteins was loaded from the brain lysate compared to the spinal cord; α-tubulin served as a loading control. (C) Spectrum of LDB3-L and LDB3-S isoforms detected by SMRT sequencing in the brain of six month old mice (n = 3). Relative percentages of total reads are shown. (D) Representative gel electrophoresis of Ldb3 splice isoforms in different mouse tissues in a RT-PCR assay (n = 6 mice). Exon composition identified by Sanger sequencing is indicated. Chromatogram of a cloned amplicon from the lower band (*) in the spinal cord validates the novel splicing events joining Ldb3 exon 3 to 6 and exon 6 to 8. Predicted reading frame including premature stop codon in exon 6 is shown. Dotted line indicates continuity of exon 6 sequence in the chromatogram. See Supplementary Table S2 for all primer sequences. (E) Representative immunofluorescence on lumbar spinal cord cross sections of six month old mice (n = 6) show staining for the exon ___domain-specific LDB3 antibodies (green) in NeuN (red)-positive neurons of the anterior horn. Scale bar: 20 µm. (F) Representative immunohistochemistry on serial cross sections of human L1-L2 spinal segments (n = 3 autopsies) show staining for the exon-specific antibodies in neurons of Clarke’s dorsal nucleus (1), the lateral column (2), and the anterior horn (3). Individual tiles were acquired at 20 × magnification and stitched together into a composite image. Original blots/gels for the image B and D are presented in Supplementary Fig. 2 of supplementary file 1.