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. 2017 Dec 14;46(4):2030–2044. doi: 10.1093/nar/gkx1255

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© The Author(s) 2017. 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 License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 2. — Impacts of the two major PCBP isoforms, PCBP1 and PCBP2, on CDK2 ex5 splicing. (A) Impact of isoform-specific depletions of PCBP1 and PCBP2 proteins on splicing of exon 5 in the endogenous CDK2 transcript. K562 cells were individually transfected with 2 distinct siRNAs that co-target PCBP1 and PCBP2 mRNAs (siRNA PCBP1/2-1, PCBP1/2-2 as described (19)). The study was done in biological triplicate or with three distinct siRNAs that selectively target PCBP1 (PCBP1-1, PCBP1-2, PCBP1-3), or PCBP2 (PCBP2-1, PCBP2-2, PCBP2-3). Left: Impact of isoform-specific PCBP depletions on cassette exon 5 splicing of the endogenous CDK2 transcript assessed by RT-PCR. Right: Quantification and statistics of the cassette exon 5 splicing data. Summary of the splicing inclusion values are shown on the histogram to the right. ^∗∗∗P < 0.001, n.s.: not significant. (B) Impacts of PCBP1 and PCBP2 depletion/repletion on CKD2 ex5 mingene splicing. The minigene (CDK2-C₁₃) plasmid was co-transfected into K562 cells along with a control siRNA (CTRL) or a siRNA co-targeting PCBP1/2 (siRNA PCBP1/2-2 as in (19)) and/or with plasmids expressing recombinant Flag-tagged PCBP1 or PCBP2 (encoded by siRNA-immune mRNAs). The experiment was carried out twice (n = 2). Left: Western blot analysis of protein expression. Labeling of lanes: V (empty vector), CP1 (Flag-PCBP1 expression vector), and CP2 (Flag-PCBP2 expression vector). Western blot assay confirmed siRNA-mediated depletion of endogenous PCBP1 or PCBP2 and expression of the recombinant Flag-PCBP proteins. Right: Impacts of depletion and repletion on CDK2 ex5 minigene splicing. The mean level of exon 5 inclusion (±SD) in each transfection is displayed below the corresponding lane (n = 2). ^∗P < 0.05, ^∗∗P < 0.01, n.s.: not significant.