Human HOIP/RNF31 Antibody

Detection of HOIP/RNF31 by Western Blot

CYLD is phosphorylated at a novel Ser568 site as well as within the phospho-rich patch to activate its activity(A) Qualitative DUB assay of CYLD (aa 436–956) purified from sf9 cells. CYLD was incubated in buffer alone, or with lambda PP as in Figures 1D and 1E. lambda PP was removed and dephosphorylated CYLD was either incubated with buffer alone (−), IKK beta, or the IKK complex (IKK beta  + NEMO) prior to DUB assay using Lys63-Ub4 as substrate. For comparison, phosphorylated CYLD from sf9 cells was included (control).(B) as in (A) but using Met1-Ub4 as substrate.(C) Purification and western blot analysis of the TNFR1 complex in U2OS/NOD2 cell lines with subsequent lambda PP treatment. Cells were stimulated with biotin-TNF for 15 min (closed circles) or after lysis (open circles).(D) Purification and western blot analysis of the TNFR1 complex in HCT116 and U937 cells expressing shRNA targeting CYLD or control shRNA. Cells were stimulated with biotin-TNF for the indicated time points.(E and F) Western blot analysis of U2OS/NOD2 cells transfected with siRNA targeting OTULIN or mismatch control (shMM) and treated or not with IKK inhibitors VII + XII (E) or TAK1 inhibitor 5Z-7-oxozeaonol (F) for 30 min before stimulation with L18-MDP for 1 h or TNF for 15 min.(G) Workflow for identification of CYLD phosphorylation sites by mass spectrometry.(H) Schematic representation of identified CYLD phosphorylation sites.(I) Analysis of evolutionary conservation of identified phosphorylation sites. TNF-induced sites are shown in red.(J) Spectral data for identification of the novel S568 site in CYLD.(K) Qualitative DUB assay with CYLD variants purified from sf9 cells. Samples were either incubated in buffer alone or in the presence of lambda PP prior to incubation with Lys63-Ub4 as substrate. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/34610306), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of HOIP/RNF31 by Western Blot

Non-phosphorylation-mediated activity of CYLD retains LUBAC at the TNF-RSC and restores normal Met1-Ub, but not Lys63-Ub, deposition(A) Purification and western blot analysis of the TNFR1 complex in U2OS/NOD2 cell lines stimulated with biotin-TNF for the indicated time points.(B) Schematic model of how Ub-binding via CAP-Gly3 and phosphorylation affects CYLD-regulation of Lys63- and Met1-Ub at receptor complexes. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/34610306), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of HOIP/RNF31 by Western Blot

The absence of CYLD activity leads to reduced LUBAC retention at the TNF-RSC as well as increased Lys63-Ub and reduced Met1-Ub on receptor components(A–C) Purification and western blot analysis of the TNFR1 complex in THP1 cells with shRNA knockdown of CYLD or mismatch control (A) and U2OS/NOD2 cell lines (B and C) stimulated with biotin-TNF as indicated.(D) UbiCRest analysis of TNFR1 complexes purified as in (B). Samples were incubated or not with OTULIN and/or OTUD1 as indicated and analyzed by western blotting. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/34610306), licensed under a CC-BY license. Not internally tested by R&D Systems.