Role of DDB1 in GRK5 Degradation Figure 6. DDB1 mediates UV irradiation-induced GRK5 degradation. (A) 293 T cells were treated with 20, 100 or 300 J/m2 of UV-C irradiation. One or two hours after UV treatment, cells were harvested and analyzed by Western blotting with antibodies specific for the indicated proteins. (B) 293 T cells were incubated for 0, 5, 15, 30, 60, or 120 min after 20 J/m2 UV-C irradiation, and then cells were harvested and analyzed by Western blotting with antibodies specific for the indicated proteins. Quantitative graphs are shown. The amount of GRK5 at 0 h was defined as 100%. Data are the mean6SE from 4 independent experiments. (C) 293 T cells were infected by lentivirus encoding control or DDB1 RNAi. After 72 h, cells were treated with 20 J/m2 UV-C irradiation, and then incubated for the indicated time. Cells were harvested and analyzed by Western blotting. (D) 293 T cells were pretreated with or without isoproterenol (1 mM) for 20 min before 20 J/m2 UV-C irradiation, and then incubated in the presence or absence of isoproterenol (1 mM) for the indicated time. Cells were harvested and analyzed by Western blotting. doi:10.1371/journal.pone.0043997.g006 buffered saline. Titers were determined by infecting 293 T cells with serial dilutions of concentrated lentivirus. GFP expression in infected cells was determined 48 h after infection, and for a typical preparation, the titer of lentivirus was approximately 108 infectious units (IFU)/ml. Isolation and Identification of GRK5-associated Proteins MDA-MB-231 cells were stably transfected with GRK5-Flag or GFP lentivirus. HUVEC cells were transiently transfected with GRK5-Flag or GFP by lentivirus. About 108 cells were harvested and lysed in IP buffer (50 mM Tris.HCl, pH 7.5, 150 mM NaCl, 0.5% NP-40, 10% glycerol, 1 mM EDTA, 10 mM NaF, plus PLOS ONE | www.plosone.org 8 August 2012 | Volume 7 | Issue 8 | e43997