e1 ubiquitin activating enzyme  (R&D Systems)

Journal: Nucleic Acids Research

Article Title: PCNA-binding activity separates RNF168 functions in DNA replication and DNA double-stranded break signaling

doi: 10.1093/nar/gkae918

Figure Lengend Snippet: PCNA ubiquitylation facilitates RNF168-binding. ( A ) ITC was conducted by titrating synthetic peptide (p21, GRKRRQTSMTDFYHSKRRLIFS-amide where underlined residues denote PIP box; syringe, 150–300 μM) into a solution of PCNA (cell; 30 μM) in TBS. Control experiments using peptide injected into buffer alone showed minimal heats with no evidence of titration. Analysis of the isotherm yielded K d = 76.3 nM and stoichiometry = 0.93 (Microcal Origin software). ( B ) Replicate plates of H1299 cells were infected with adenovirus vectors encoding FLAG-RNF168 WT and HA-PCNA (or an ‘empty’ adenovirus vector for control). Thirty-six hours post-infection, chromatin extracts were prepared, normalized for protein content and immunoprecipitated with anti-FLAG antibody in the presence of different concentrations of peptides corresponding to the p21 or Polη PIP boxes. Anti-FLAG immunoprecipitates were resolved on SDS-PAGE, transferred to nitrocellulose and analyzed by immunoblotting with the indicated antibodies. ( C ) Replicate plates of H1299 cells were co-infected with adenovirus vectors encoding HA-PCNA and FLAG-RNF168 WT or HA-PCNA and RNF168 ΔDPIP. Some cells received an ‘empty’ adenovirus vector instead of HA-PCNA (for control). Thirty-six hours post-infection, some plates were treated with 2 mM HU for 2 h. Chromatin extracts were prepared, normalized for protein content and immunoprecipitated with anti-HA antibody in the presence or absence of the p21 PIP box peptide (1 mM). Anti-HA immune complexes were resolved on SDS-PAGE, transferred to nitrocellulose and analyzed by immunoblotting with the indicated antibodies. ( D ) Replicate plates of H1299 cells were infected with adenovirus vectors encoding wild-type PCNA (HA-PCNA WT), ubiquitylation-resistant PCNA (HA-PCNA K164R) or a PCNA-ubiquitin fusion (HA-PCNA-Ub) in combination with FLAG-RNF168 WT adenovirus. Some cells received an ‘empty’ adenovirus vector instead of HA-PCNA viruses(for control). Thirty-six hours post-infection, chromatin extracts were prepared, normalized for protein content and immunoprecipitated with anti-HA antibody. Anti-HA immune complexes were resolved on SDS-PAGE, transferred to nitrocellulose and analyzed by immunoblotting with the indicated antibodies. ( E ) Replicate plates of H1299 cells were transfected with expression plasmids encoding WT or mutant forms of RNF168. The transfected cultures were then infected with adenovirus vector encoding wild-type PCNA (HA-PCNA WT) or with an ‘empty’ adenovirus vector (for control). Thirty-six hours post-infection, chromatin extracts were prepared, normalized for protein content and immunoprecipitated with anti-HA antibody. Anti-HA immune complexes were resolved on SDS-PAGE, transferred to nitrocellulose and analyzed by immunoblotting with the indicated antibodies.

Article Snippet: Ubiquitylation assays were performed in 25 μl reactions in which the components were added in the following order: dd H2O, 1× Energy regeneration solution (#B-10 R&D systems), 75 mM ubiquitin (#U-100H R&D systems), 1.6 mM FLAG-PCNA substrate (expressed and purified in bacteria), 0.1 mM E1 Ubiquitin Activating Enzyme (#E-304 R&D systems), 0.2 mM E2 conjugase (UbcH5c, #E2-627 R&D systems or RAD6 #E2-613 R&D Systems), 0.2 mM E3 ligase (recombinant bacterial RAD18-RAD6 complex or RNF168 both purified in-house).

Techniques: Binding Assay, Control, Injection, Titration, Software, Infection, Plasmid Preparation, Immunoprecipitation, SDS Page, Western Blot, Transfection, Expressing, Mutagenesis