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Rbfox1 Downregulation and Altered Calpain 3 Splicing by FRG1 in a Mouse Model of Facioscapulohumeral Muscular Dystrophy (FSHD)

Figure 8

Rbfox1 down-regulation is responsible for significant portion of the splicing alterations in FRG1 mice.

(a) Specific Rbfox1 knockdown was confirmed by real-time RT-PCR and immunoblotting using RNAs and proteins isolated from C2C12 muscle cells expressing a control non-silencing shRNA or an shRNA specific for Rbfox1 (shRNA#1). (b) Examples of alternative splicing changes caused by Rbfox1 knockdown are showed. Numbers are the percentage of exon inclusion. Black boxes illustrate constitutive exons, white boxes alternatively spliced exons. (c) Rbfox1 overexpression causes alternative splicing changes opposite to FRG1 over-expression. Specific Rbfox1 over-expression was confirmed by real-time RT-PCR and immunoblotting using RNAs and proteins isolated from C2C12 muscle cells expressing an empty vector (EV) or a Myc-tagged Rbfox1 (F1) either in proliferating or differentiating C2C12 muscle cells. (d) Examples of alternative splicing changes caused by Rbfox1 over-expression are showed. Black boxes illustrate constitutive exons, white boxes alternatively spliced exons. (e) Selective in vivo association of Rbfox1 to target regions displaying putative Fox binding sites (FBS). RIP experiment on samples from (c) using anti-Myc or control IgG antibodies. Immunoprecipitated material was analyzed by RT-PCR, quantified using the Typhoon, normalized versus the relative input and plotted as fold enrichment versus the IgG. RT-minus control experiments showed the absence of DNA contamination (data not shown).

Figure 8

doi: https://doi.org/10.1371/journal.pgen.1003186.g008