Supplemental Data UV-Induced Ubiquitylation of XPC Protein Mediated by UV-DDB-Ubiquitin Ligase Complex

Preparation of Cell Lysates For immunoblot analyses of NER factors from human fibroblast cells, the cells in 60-mm dishes were washed twice with phosphate-buffered saline and lysed in situ with 0.25 ml of ice-cold NP lysis buffer [25 mM Tris-HCl (pH 8.0), 1 mM EDTA, 10% glycerol, 1% Nonidet P-40, 10 mM N-ethylmaleimide, 0.25 mM phenylmethylsulfonyl fluoride (PMSF), a protease inhibitor cocktail (Complete, Roche Diagnostics)] containing 0.3 M NaCl. After incubation on ice for 30 min, the cell lysates were scraped into microfuge tubes and centrifuged for 10 min at 20,000 × g to obtain the supernatant fractions. The resulting pellets were resuspended with the aid of sonication in the same volume of NP lysis buffer containing 0.3 M NaCl. Protein concentrations of the supernatant fractions were determined according to the method of Schaffner and Weissmann (1973) using bovine serum albumin (BSA) as a standard.