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. 2017 Aug 11;6:e27192. doi: 10.7554/eLife.27192

Figure 8. Redirected calcineurin A splicing in differentiated C2C12 myotubes.

(A) RT-PCR of redirected splicing of calcineurin A events in differentiated C2C12 cells. Two of the four calcineurin A splicing events are present in C2C12. For calcineurin A ASO, 9 μM Ppp3ca ex13 3’ss and 15 μM Ppp3cb ex13 5’ss morphlinos were delivered, and for control ASO, 24 μM standard control morpholino were delivered. (B) Immunofluorescence of Nfatc3 in differentiated C2C12 after morpholino delivery. (C) mRNA levels of Nfatc targets after knockdown of Nfatc3 in differentiated C2C12 cells. Relative mRNA levels are standardized to Mrps7 mRNA levels; single asterisk (*) denotes p<0.05 significance by student T-test, n = 3 biological replicates. Displayed are mean with SD bars.

Figure 8.

Figure 8—figure supplement 1. Analysis of calcineurin A splicing during C2C12 differentiaion, Nfatc1 and Nfatc2 protein localization in C2C12, and validation of Nfatc3 antibody.

Figure 8—figure supplement 1.

(A) PSI for calcineurin A splicing events from undifferentiated (U) and days in after differentiation media was added days 1 through 4 (D1, D2, D3, D4). (B) Localization of Nfatc1 and Nfatc2 by immunofluorescence in differentiated C2C12 with ASOs. (C) Knockdown of Nfatc3 using silencer siRNAs to confirm signal of Nfatc3 antibody.
Figure 8—figure supplement 2. Sequences for antisense morpholinos and primers.

Figure 8—figure supplement 2.

(A) Morpholino sequences used for redirected splicing. (B) Primers used for splicing, genotyping, and mRNA levels.