Role of DDB1 in GRK5 Degradation Figure 5. DDB1 regulates GRK5 degradation. (A) 293 T cells were infected with lentivirus expressing control RNAi or DDB1 RNAi. Cells were treated with 100 mg/ml CHX for the indicated time. Cell lysates were 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. *p,0.01. (two-way ANOVA) compared with control RNAi. (B) 293 T cells were treated with geldanamycin at indicated concentration for 24 h at 37uC. Cell lysates were analyzed by Western blotting with antibodies specific for the indicated proteins. (C) 293 T cells were pretreated with or without 10 mM MG132 for 1 h followed by incubation with geldanamycin for 12 h. Cell lysates were analyzed as in B. (D) 293 T cells were infected by lentivirus encoding control or DDB1 RNAi. Cells were treated with 1 mM geldanamycin for the indicated time. Cell lysates were analyzed as in B. doi:10.1371/journal.pone.0043997.g005 were from Invitrogen. Rabbit anti-DDB2 antibody, rabbit anti- GRK2 antibody, and protein A/G agarose were from Santa Cruz Biotechnology. Rabbit anti-CUL4B antibody, rabbit anti-b-actin antibody, mouse antibody against FLAG epitope, rabbit antibody against HA epitope, mouse IgG, anti-Flag M2 affinity gel, and anti-HA agarose were from Sigma. Goat anti-GRK5 antibody was from R&D. Geldanamycin and cycloheximide (CHX) were from Sigma. Plasmid Construction Plasmids encoding tagged bovine GRK2 and GRK5, and human GRK6 were constructed by PCR mutagenesis. Construc- tion of shRNA plasmids for human DDB1, human CUL4A, and human CUL4B was performed as described [53]. LacZ shRNA was used as a control. The shRNA lentivirus system, which uses the FG12 and package vector to enable simultaneous expression of the GFP protein and shRNA were obtained from Dr. Gang Pei (Chinese Academy of Sciences, Shanghai, China). Sequences for DDB1 RNAi are: GCG AGA GCA TTG ACA TCA TTA (sense) and TAA TGA TGT CAA TGC TCT CGC (antisense). CUL4A: GCA GGA CCA CTG CAG ACA AAT (sense) and ATT TGT CTG CAG TGG TCC TGC (antisense). CUL4B: GCC ACG TAC CGA TAC AGA AGA (sense) and TCT TCT GTA TCG GTA CGT GGC (antisense). The plasmids DDB1-Flag (Addgene plasmid 19918), CUL4A-HA (Addgene plasmid 19907), and Myc- CUL4A (Addgene plasmid 19951) were gifts of Dr. Yue Xiong. The plasmid DDB1 was constructed into pcDNA3.0. Cell Culture and Plasmid Transfection 293 T cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% FBS. Cells were seeded in 60 or 100 mm tissue culture dishes at 0.6–26106/dish 20 h before transfection and transfected plasmid using calcium phosphate/DNA co- precipitation method or infected by lentivirus. Assays were performed 48 h (for expressing experiments) or 72 h (for RNAi experiments) after transfection. MDA-MB-231 cells stably expres- sing GFP or GRK5-Flag were cultured in Leibovitz’s L-15 plus 10% FBS. For protein decay analysis, 293 T cells were treated with CHX at 100 mg/ml. Generation and Titer of Lentivirus The sequences predicted to target the human DDB1 or CUL4A were used for the construction of shRNA lentivirus. FG12-hU6- shRNA and packaging vectors were co-transfected into 293 T cells and the resulting supernatant were collected after 48 h. Virus was recovered after ultracentrifugation resuspension in phosphate- PLOS ONE | www.plosone.org 7 August 2012 | Volume 7 | Issue 8 | e43997