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. 2018 May 16;46(12):6069–6086. doi: 10.1093/nar/gky401

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© The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Figure 6. — MBNL1 protein regulates 11 out of 14 DASEs during monocyte-to-macrophage differentiation. (A) Quantitative real-time RT-PCR shows effective knockdown of MBNL1 mRNA by dsi1-MBNL1 nucleofection and sh2-MBNL1 transduction (left), and knockdown of RBFOX2 mRNA by dsi2-RBFOX2 nucleofection and sh1-RBFOx2 transduction (right), all in THP-1. Knockdown of MBNL1 or RBFOX2 has no major effect on one another's mRNA. (B) Western blotting shows MBNL1 protein downregulation by sh2-MBNL1 and slightly by dsi1-MBNL1, as well as RBFOX2 downregulation by both sh1-RBFOX2 and dsi2-RBFOX2. Each knockdown did not affect the other protein with the exception of sh2-MBNL1 cells showing some RBFOX2 down-regulation, which was not seen in dsi1-MBNL1 and shRNA control. Lane labels at the bottom. (C) Radioactive RT-PCR showing that MBNL1 knockdown in THP-1 cells changed 10 DASEs (green labels) in the same direction as PMA3D treatment, while both MBNL1 and RBFOX2 knockdowns in PMA3D cells changed the DASE of GOLIM4 (red) in the expected direction. AS type indicated in parenthesis, including cassette exons (CA) and alternative 5′ splice sites (AD). Bottom right corner, mini-table summary of the 14 DASE changes upon SF knockdown. (D) Western blotting analysis of RNA pulldowns with 139-nt probes for NCOR2 (probes 1–3), LIRRFIP2 (probes 1–4), and GOLIM4 (probe 1), numbered from 5′ to 3′ (Supplementary Figure S8). Wild-type probes on the left and mutants on the right. FT, flow-through.