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Fig. 8 | Molecular Neurodegeneration

Fig. 8

From: Cytoplasmic mislocalization of RNA splicing factors and aberrant neuronal gene splicing in TDP-43 transgenic pig brain

Fig. 8

Mutant TDP-43 causes abnormal splicing of NMHC II-B and promotes NMHC II-B degradation. a Diagram of the mouse NMHC II-B gene from exon 5 to exon 9. The exons are shown as boxes and the lines between exons are introns. The IDDE sequence is the NeuN binding region, and the alternative splicing exon N30 is indicated as red box between exon 5 and exon 6. The P5’ and P3’ represent the PCR primers to identify N30 inclusion (+N30) or exclusion (−N30). b RT-PCR gel result (upper panel) and quantification (lower panel) of the inclusion (+N30) and exclusion (−N30) of exon N30 in the brain cortex of non-transgenic and transgenic TDP-43 pigs (TG-5, −7, −11, −13, −15, −17, −23, −25). GAPDH served as a control. Relative +N30 and -N30 isoform levels are shown below each lane and are normalized by the total NMHC II-B gene amount. c Western blot analysis is unable to distinguish +N30 and –N30 because of their few amino acid differences but shows a decrease of NMHC II-B in homogenates, cytoplasmic, and nuclear fractions from transgenic TDP-43 pig cortex compared with non-transgenic (C) pig. GAPDH and TFIID are cytoplasmic and nuclear marker proteins, respectively. The relative levels of NMHC II-B, NeuN, PSF, and TDP-43 in the homogenates (Hom) (ratios of homogenate proteins to GAPDH) as well as cytosolic (Cyto) (ratios of cytoplasmic proteins to GAPDH) and nuclear (ratios of nuclear proteins to TFIID) fractions are presented beneath the blot. The data are mean ± SE (n = 3 animals per group). *p < 0.05; **p < 0.01 compared with non-Tg. d Reduced NMHC II-B immunostaining in the motor neurons in the spinal cord of transgenic TDP-43 (TG) pig compared with non-Tg (Control) pig. Two representative images are shown. Scale bar: 50 μm

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