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. 2022 Jul 12;12(9):2158–2179. doi: 10.1158/2159-8290.CD-21-0205

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©2022 The Authors; Published by the American Association for Cancer Research

This open access article is distributed under the Creative Commons Attribution 4.0 International (CC BY 4.0) license.

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Figure 4. — RNF113A is a phosphoprotein and its methylation repels the phosphatase PP4. A, SILAC quantitative proteomics analysis of proteins that interact with RNF113A K20me0 and RNF113A K20me3 peptides. Data represent two independent experiments (forward and reverse directions). Proteins are plotted by their SILAC ratios in the forward (x-axis) and reverse (y-axis) experiments. Specific interactors of RNF113A K20me0 reside in the lower left quadrant. The four PP4 complex subunits are circled in blue. L/H, light over heavy fraction ratio. B, 293T cell extracts ectopically expressing HA-tagged PPP4R3a and PPP4c subunits were used for pulldowns with the indicated RNF113A peptides, followed by immunoblot analysis using the indicated antibodies. C, Immunoblot analysis of endogenous PPP4R3A following pulldowns with indicated RNF113A peptides using SCLC DMS-114 cell extract. D, Immunoblot analysis of recombinant PPP4R3A following pulldowns with the indicated RNF113A peptides. E, Immunoblot analysis of endogenous PPP4R3A pulldown using GST labeled recombinant RNF113A WT, K20A, K20R, and K20F mutants. F, Phosphorylation-dependent mobility shift of RNF113A on SDS-PAGE immunoblotting (indicated by arrows). HeLa cell extracts were treated with λ phosphatase (λ PPase), FastAP thermosensitive alkaline phosphatase (Fast AP), or calf intestinal alkaline phosphatase (CIP). Ku80 was used as a loading control. G, Identification of potential RNF113A phosphorylation sites based on the Phosphosite Plus references (y-axis) and confirmed by two independent mass spectrometry analyses (underlined residues; see also Supplementary Table S4). The schematic shows the sequence surrounding the methylated K20 and PPP4R3a binding motif (FxxP). Summary of phosphorylation and methylation site mutants of RNF113A generated in this study (bottom). H, Immunoblot confirmation of phosphorylation-dependent mobility shift of the indicated RNF113A mutants expressed in HeLa cells with or without CIP treatment. Ku80 was used as a loading control. I, Immunoblot analysis of RNF113A dephosphorylation assays using HA-RNF113A purified from HeLa cells, with either FastAP or PP4 phosphatases treatment followed by immunoblot analysis using a phospho-CDK-consensus motif antibody. In all panels, representative of at least three independent experiments is shown unless stated otherwise. The numbers below the immunoblot lines represent the relative signal quantification (see also Supplementary Table S5).