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. 2020 Nov 2;48(20):11626–11644. doi: 10.1093/nar/gkaa814

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© The Author(s) 2020. 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 8. — RNA distribution in polysome fractions obtained from cells overexpressing SINEUP RBPs. (A) Representative polysome gradient profile with optical density (290 nm). Fractions and correspond to the 15% and 45% sucrose fractions, respectively. (B) Polysome profiling of EGFP mRNA (green) and SINEUP-GFP RNA (blue). RNA distribution was quantified by RT-qPCR. Each fraction of EGFP mRNA in cells co-transfected with EGFP and SINEUP-GFP vectors (d–f) was compared with the corresponding fraction in cells transfected with EGFP vector alone (a–c). *P < 0.05, **P < 0.01 by Student's t-test. Data are means ± SD from three independent experiments. Fractions of SINEUP RNA in cells co-transfected with EGFP and SINEUP-GFP vectors are shown in g-i, and those in cells transfected with SINEUP RNA alone are shown in m. The protein distribution in polysome fractions with non-/overexpressed PTBP1 or HNRNPK were tested (j-m). Equal volumes of solution were pooled as Free/40S (Free or 40S binding RNA fraction from – in A), Mono (monosome fraction from –), Light (light polysome fraction from –), and Heavy (Heavy polysome fraction from –) and were applied to 10% SDS gels to detect each target protein. Western blotting images representative of least three independent experiments are shown.

Inline graphic — RNA distribution in polysome fractions obtained from cells overexpressing SINEUP RBPs. (A) Representative polysome gradient profile with optical density (290 nm). Fractions and correspond to the 15% and 45% sucrose fractions, respectively. (B) Polysome profiling of EGFP mRNA (green) and SINEUP-GFP RNA (blue). RNA distribution was quantified by RT-qPCR. Each fraction of EGFP mRNA in cells co-transfected with EGFP and SINEUP-GFP vectors (d–f) was compared with the corresponding fraction in cells transfected with EGFP vector alone (a–c). *P < 0.05, **P < 0.01 by Student's t-test. Data are means ± SD from three independent experiments. Fractions of SINEUP RNA in cells co-transfected with EGFP and SINEUP-GFP vectors are shown in g-i, and those in cells transfected with SINEUP RNA alone are shown in m. The protein distribution in polysome fractions with non-/overexpressed PTBP1 or HNRNPK were tested (j-m). Equal volumes of solution were pooled as Free/40S (Free or 40S binding RNA fraction from – in A), Mono (monosome fraction from –), Light (light polysome fraction from –), and Heavy (Heavy polysome fraction from –) and were applied to 10% SDS gels to detect each target protein. Western blotting images representative of least three independent experiments are shown.