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. 2019 Feb 4;47(8):4011–4025. doi: 10.1093/nar/gkz055

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© The Author(s) 2019. 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 3. — Quantitative interaction proteomics reveal UVSSA interaction partners and required UVSSA-domains. (A) Scatter plot of log₂ SILAC ratios of proteins isolated by GFP-pulldown in UV^SS-A cells stably expressing either GFP-UVSSA or GFP (non-specific binding control). The experiment was conducted in duplicate with a label swap. The log₂ SILAC ratios of proteins identified in the forward experiment (GFP-UVSSA versus GFP, H/L, x-axis) are plotted against the log₂ SILAC ratios of proteins identified in the reversed experiment (GFP-UVSSA versus GFP, L/H, y-axis). Proteins were classified as specific UVSSA interactors (marked in blue) when log₂ SILAC ratio >0.6 (indicated by gray dotted line) in both replicates. (B) GO-term analysis of the 66 proteins identified as specific interactors of UVSSA. A selection of the top 10 enriched biological process pathways is shown. GC: gene count; FDR: false discovery rate. (C) Scatter plot of log₂ SILAC ratios of proteins identified in the GFP-pulldowns of wt UVSSA versus ΔVHS, only proteins that were also identified in the GFP-UVSSA versus GFP proteomics experiment are depicted. The experiment was conducted in duplicate, including a label swap. The log₂ SILAC ratios of proteins identified in the forward experiment (wt versus ΔVHS, H/L, x-axis) are plotted against the log₂ SILAC ratio of proteins identified in the reversed experiment (wt versus ΔVHS, L/H, y-axis). The majority of proteins have similar binding ability to the ΔVHS mutant compared to the wt (log₂ SILAC ratio <0.6, proteins marked in gray). Proteins marked in blue represent proteins whose interaction with UVSSA is decreased in the absence of the VHS domain. (D) Scatter plot of log₂ SILAC ratios of proteins identified in the GFP-pulldowns of wt UVSSA versus ΔDUF only proteins that were also identified in the GFP-UVSSA versus GFP proteomics experiment are depicted. The experiment was conducted in duplicate, including a label swap. The log₂ SILAC ratios of proteins identified in the forward experiment (wt versus ΔDUF, H/L, x-axis) are plotted against the log₂ SILAC ratio of proteins identified in the reversed experiment (wt versus ΔDUF, L/H, y-axis). Proteins marked in blue have a reduced interaction with UVSSAΔDUF compared to wt (proteins are marked in blue, log₂ SILAC ratio >0.6, gray dotted line marks the threshold). (E) Cross-linked nuclear extracts of UV^SS-A patient cell line (TA24), stably expressing the indicated constructs were subjected to GFP immunoprecipitation. Non-complemented UV^SS-A patient cell line (-) was used as negative binding control. WCE: whole-cell extract, IP: Immunoprecipitation. Western blot analysis of the co-immunoprecipitated proteins was performed for GFP, Spt16, SSRP1, USP7 and CSA.