Figure 3. H2A ubiquitylation after UV irradiation is performed by the UV–RING1B complex. (A) Protein interaction partners of RING1B and DDB2. Mass spectrometry analysis after sequential immunoprecipitations with FLAG and RING1B antibodies revealed DDB1 and CUL4B as main interaction partners of DDB2 and RING1B. A comprehensive list of the identified unique peptides after RING1B and control immunoprecipitations (with or without UV irradiation) is provided in Table S5. (B) Assembly of the UV–RING1B complex. Plasmids expressing FLAGDDB1, FLAGDDB2, and FLAGRING1B were cotransfected in combination with either control plasmid or a plasmid encoding FLAG-STREPCUL4B. After immunoprecipitation with STREP-Tactin beads, the purified material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 5%. (C) Visualization of the UV–RING1B complex. Purified UV–RING1B complex was subjected to SDS gel electrophoresis and colloidal Coomassie staining. Mass spectrometry analysis revealed the presence of all four subunits (bold). A comprehensive list of unique peptides is provided in Table S6. (D) The UV–RING1B complex catalyzes ubiquitylation of H2A in vitro. Ubiquitylation assays were performed with recombinant H2A, E1 (UBA1), E2 (UBCH5), and either GST (control) or the UV–RING1B complex. Reactions were performed at 37°C, and samples were taken at the indicated time points. Material of the respective time points was subjected to Western blotting and probed with the indicated antibodies. (E) The UV–RING1B complex catalyzes monoubiquitylation of nucleosomal H2A. Ubiquitylation assays were performed with recombinant nucleosomes, E1 (UBA1), E2 (UBCH5), and either GST (control) or UV-RING1B complex. Reactions lacking E1 (−E1) were performed as additional controls. The ubiquitylation assays were performed at 37°C for 5 h, and samples or pure substrate (Substrate) were subjected to Western blotting and probed with H2A antibodies.
Figure 3.

H2A ubiquitylation after UV irradiation is performed by the UV–RING1B complex. (A) Protein interaction partners of RING1B and DDB2. Mass spectrometry analysis after sequential immunoprecipitations with FLAG and RING1B antibodies revealed DDB1 and CUL4B as main interaction partners of DDB2 and RING1B. A comprehensive list of the identified unique peptides after RING1B and control immunoprecipitations (with or without UV irradiation) is provided in Table S5. (B) Assembly of the UV–RING1B complex. Plasmids expressing FLAGDDB1, FLAGDDB2, and FLAGRING1B were cotransfected in combination with either control plasmid or a plasmid encoding FLAG-STREPCUL4B. After immunoprecipitation with STREP-Tactin beads, the purified material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 5%. (C) Visualization of the UV–RING1B complex. Purified UV–RING1B complex was subjected to SDS gel electrophoresis and colloidal Coomassie staining. Mass spectrometry analysis revealed the presence of all four subunits (bold). A comprehensive list of unique peptides is provided in Table S6. (D) The UV–RING1B complex catalyzes ubiquitylation of H2A in vitro. Ubiquitylation assays were performed with recombinant H2A, E1 (UBA1), E2 (UBCH5), and either GST (control) or the UV–RING1B complex. Reactions were performed at 37°C, and samples were taken at the indicated time points. Material of the respective time points was subjected to Western blotting and probed with the indicated antibodies. (E) The UV–RING1B complex catalyzes monoubiquitylation of nucleosomal H2A. Ubiquitylation assays were performed with recombinant nucleosomes, E1 (UBA1), E2 (UBCH5), and either GST (control) or UV-RING1B complex. Reactions lacking E1 (−E1) were performed as additional controls. The ubiquitylation assays were performed at 37°C for 5 h, and samples or pure substrate (Substrate) were subjected to Western blotting and probed with H2A antibodies.

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