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. 2023 Sep 21;14:5868. doi: 10.1038/s41467-023-41284-9

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — AGPC acidic guanidinium thiocyanate–phenol-chloroform, I INP, L LEAP-RBP, SILAC SILAC LC–MS/MS analysis, SRA SRA analysis, E* significantly UV-enriched*, NE not E*. a Schematic illustration of the experimental approach. Heavy SILAC-labeled UV-crosslinked and light SILAC-labeled non-crosslinked cells were mixed prior to repeated AGPC extraction and isolation of RNP fractions by L or I methods. Graphic prepared in BioRender. b Volcano plots showing proteins identified as E* (red) or NE (blue) in I or L fractions by SILAC. Log₂(CL/nCL) ratios were generated with SPI_CL values and average SPI_nCL values. Tested against the null hypothesis that protein log₂(CL/nCL) ratios (n = 3 biologically independent samples) are equal to 0 using unpaired, upper-tailed, heteroscedastic t tests (RNA-binding proteins are expected to be recovered from UV-crosslinked cells in greater amounts). Correction for multiple hypothesis testing was performed using the Benjamini–Hochberg approach and a false-discovery rate of 5%. c GO-enrichment analysis of proteins identified as E* in I or L fractions by SILAC (Fisher’s Exact, two-tailed, correction for multiple hypothesis testing performed using the Benjamini–Hochberg approach and a false-discovery rate of 5%). d Venn diagram showing overlap of total and E* proteins identified in I and/or L fractions by SILAC. Pie charts showing the number of RBPs and non-RBPs identified as E* or NE in I or L fractions by SILAC. For information on protein ID assignment and protein lists for each category or GO term used in this study, see Supplementary Data 6 and 7. e Stacked bar charts showing the number of proteins with annotated RNA-binding or RNA-related functions identified as E* or NE in I or L fractions by SILAC. f Histograms showing average log₂(CL/nCL) ratios of RBPs and non-RBPs identified in I or L fractions by SILAC. Font color for individual proteins reflects conclusions from SRA and immunoblot analysis of total clRNP fractions; red text: RNase-sensitive RBP; blue text: undetected or RNase-insensitive protein. Asterisks: E*. I and L SILAC LC–MS/MS experiments (a) were performed once with three biologically independent samples for each SILAC label group (CL, nCL). Source data are provided as Source Data file.