uv c irradiation  (Philips Healthcare)

Journal: Nucleic Acids Research

Article Title: The interaction of XPG with TFIIH through p62 and XPD is required for the completion of nucleotide excision repair

doi: 10.1093/nar/gkag078

Figure Lengend Snippet: The p62- and XPD-interacting domains of XPG additively contribute to NER activity. ( A ) The XPG map with the p62 interaction domains (blue) and the XPD interaction domains (orange) highlighted. ( B ) Western blot of cells expressing XPG-∆p62, XPG-∆XPD, and XPG-∆p62/∆XPD generated by lentiviral transduction and clonal selection. XPG expression levels in each mutant cell were normalized to Ku80 expression and then further normalized to the XPG WT band. ( C ) Western blot of the input and immunoprecipitation fractions of HA-XPG. XPG, XPD, p62, and α-tubulin antibody were used for detection. A representative figure from three independent experiments is shown. Cells were collected 30 min after 5 J m −2 UV-C irradiation or non-irradiation. ( D ) Survival rate from clonogenic assays. Cells were treated with 0, 1, 2, and 4 J m −2 of UV-C, grown for 10 days, stained with methylene blue, and colonies were counted. Survival rates were normalized to the number of colonies of non-treated cells and are represented by the mean ± SEM of two independent experiments. ( E ) Kinetics of (6-4) PP repair determined by slot-blot assays. Cells were irradiated with UV-C (5 J m −2 ), genomic DNA was isolated at the indicated time points, and adduct levels were determined using an anti-(6-4) PP antibody. For the quantification, band intensities were normalized to the WT band at 0 h and are shown as the mean ± SEM of three independent experiments. ( F ) Kinetics of CPD repair determined by slot-blot assays. Cells were irradiated with UV-C (5 J m −2 ), genomic DNA was isolated at the indicated time points, and the adduct levels were determined using an anti-CPD antibody. For the quantification, band intensities were normalized to the WT band at 0 h, and are shown as the mean ± SEM of three independent experiments. ( G ) In vitro NER activity of p62- and XPD-interaction mutants of XPG. A plasmid containing a site-specific 1,3-GTG-cisplatin lesion was incubated with purified WT or mutant XPG (27 nM), XPC-RAD23B (5 nM), TFIIH (10 nM), RPA (42 nM), XPA (20 nM), and XPF–ERCC1 (13 nM) proteins for 0–90 min. The excision products were detected by annealing to a complementary oligonucleotide with a 4-dG overhang, which was used as a template for a fill-in reaction with [α- 32 P]dCTP. ( H ) Quantification of (G). Band intensities were normalized to the product of the XPG-WT reaction at 90 min. The graph is the average product formed from two independent experiments.

Article Snippet: Following 5 J m −2 UV-C irradiation, cells were further incubated with 4 mM HU and 40 μM AraC at 37°C for 0, 1, 2, and 4 h. CometChip gels were then covered with 1% LM Agarose (Trevigen, 4250-050-02) and incubated with lysis buffer (Trevigen, 4250-050-01) overnight at 4°C.

Techniques: Activity Assay, Western Blot, Expressing, Generated, Transduction, Selection, Mutagenesis, Immunoprecipitation, Irradiation, Staining, Dot Blot, Isolation, In Vitro, Plasmid Preparation, Incubation, Purification