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. 2004 Feb 18;32(3):1232–1241. doi: 10.1093/nar/gkh275

Figure 3.

Figure 3

Characterization of a CELF4 dominant-negative mutant. (A) Co-expression of the series of CELF4 N-terminal deletion mutants with the RTB33.51 cTNT minigene in primary skeletal muscle cultures. In the absence of co-expressed protein, this minigene shows activated inclusion to 70–75% which is MSE-dependent (25, lane 1). CELF4 Δ5.3 and Δ5.4 inhibited the MSE-dependent activation in muscle cultures while other deletion mutants had no effect. These RT–PCR results are representative of five independent experiments. (B) CELF4 Δ5.3 inhibits activation by CELF4 (+48). Western blot analysis using AntiXpress antibody from a parallel transfection showed that overexpression of CELF4 Δ5.3 did not affect expression of cotransfected CELF4 (+48) plasmid. (C) CELF4 Δ5.3 does not affect modulation of splicing by non-CELF splicing factors on non-MSE pre-mRNA substrates. Co-expression of an SRp20 expression vector with a minigene containing clathrin light exons 4–6 in QT35 fibroblasts induces skipping of the alternatively spliced EN exon. Co- expression of CELF4 Δ5.3 has no effect on this modulation by SRp20. SRp20 protein was detected using mAb104 monoclonal antibody and CELF Δ5.3 was detected using Anti-Xpress (data not shown).