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. 2021 Nov 25;49(21):12284–12305. doi: 10.1093/nar/gkab1120

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© The Author(s) 2021. 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 (https://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 6. — Yb1 shows preferential interaction with the full-length isoform of hnRNP R. (A, B) Volcano plots showing proteins identified by mass spectrometry that were significantly enriched after immunoprecipitation with a C-terminal-specific hnRNP R antibody relative to IgG control immunoprecipitations. Experiments were carried out in the absence (A) or presence (B) of Benzonase. Log-transformed p-values (t test) associated with individual proteins were plotted against log-transformed fold changes in protein enrichment between hnRNP R and control immunoprecipitations. (C) Anti-HA immunoprecipitation from HEK293TN cells transfected with plasmids for expression of HA-tagged hnRNP R isoforms or deletion mutants, or HA-tagged EGFP, or from untransfected cells (control). Proteins were analyzed by western blot with antibodies against Yb1 and HA-tag. In, input; IP, immunoprecipitation. Input represents 5% of the lysate used for immunoprecipitation. (D) Quantification of Yb1 co-immunoprecipitation with hnRNP R constructs. Data are mean ± SD (n = 5 independent experiments). Statistical analysis was performed using two-way ANOVA followed by Bonferroni post-hoc test; **P ≤ 0.01. (E, F) Immunoprecipitation of Yb1 from lysates of non-irradiated (0 Gy) or irradiated (9 Gy) NSC-34 cells in the absence (E) or presence (F) of Benzonase treatment. Proteins were analyzed by Western blot with antibodies against Yb1 and hnRNP R. In, input; IP, immunoprecipitation. Input represents 5% of the lysate used for immunoprecipitation. (G) Quantification of hnRNP R isoforms co-immunoprecipitating with Yb1 in the presence of Benzonase (F) normalized to the input. Data are mean ± SD (n = 3 independent experiments). Statistical analysis was performed using two-way ANOVA followed by Bonferroni post-hoc test; **P ≤ 0.01. (H, I) Scatter plots showing proteins identified by mass spectrometry after immunoprecipitation with anti-HA antibody from HEK293TN transfected with plasmids for expression of hnRNP R-FL-HA, hnRNP R-ΔN-HA or EGFP-HA as control. Data are shown as log-transformed fold changes in protein enrichment between hnRNP R-FL-HA or hnRNP R-ΔN-HA and EGFP-HA immunoprecipitations, carried out in the absence (H) or presence (I) of Benzonase. (J, K) Scatter plots showing proteins identified by mass spectrometry after immunoprecipitation with anti-HA antibody from HEK293TN expressing hnRNP R-FL-HA (J), hnRNP R-ΔN-HA (K) or EGFP-HA under non-irradiated (0 Gy) or after exposure to irradiation (9 Gy). Experiments were carried out in the absence of Benzonase. Log-transformed fold changes in protein enrichment in EGFP-HA immunoprecipitations under non-irradiated (0 Gy) and irradiated (9 Gy) conditions were plotted against log-transformed fold changes in protein enrichment in hnRNP R-FL-HA (J) or hnRNP R-ΔN-HA (K) immunoprecipitations under non-irradiated (0 Gy) and irradiated (9 Gy) conditions. Functional annotations of proteins are colour-coded. (L, M) Same as (J, K) but in the presence of Benzonase.