10 mg/ml aprotinin, 10 mg/ml benzamidine, and 0.2 mM PMSF) for 2 h. The lysate was centrifuged and the supernatant was incubated with anti-FLAG M2 affinity gel at 4uC for 8 h. The beads were subsequently washed. Bound proteins were eluted by addition of SDS sample buffer. The resulting protein complexes were electrophoresed on a 4–20% gradient polyacrylamide gel and revealed by staining with Coomassie Blue. Six selected bands were excised from the gel and analyzed by mass spectrometry. The corresponding GFP control lane were also excised and analyzed by mass spectrometry. The mass spectrometry analysis was performed at the Shanghai Applied Protein Technology Co.Ltd. LC-MS/MS Gel pieces were destained with 30% ACN/ 100 mM NH4HCO3 and dried in a vacuum centrifuge. The in- gel proteins were reduced with dithiothreitol (10 mM DTT/ 100 mM NH4HCO3 ) for 30 minutes at 56uC, then alkylated with iodoacetamide (50 mM IAA/100 mM NH4HCO3) in the dark at room temperature for 30 minutes. Gel pieces were briefly rinsed with 100 mM NH4HCO3 and ACN, respectively. Gel pieces were digested overnight in 12.5 ng/mL trypsin in 25 mM NH4HCO3. The peptides were extracted three times with 60% ACN/0.1% TFA. The extracts were pooled and dried. EttanTM MDLC system (GE Healthcare) was applied for desalting and separation of tryptic peptides mixtures. In this system, samples were desalted on RP trap columns (Agilent Technologies), and then separated on a RP column (Column technology Inc.). Mobile phase A (0.1% formic acid in HPLC-grade water) and the mobile phase B (0.1% formic acid in acetonitrile) were selected. 20 mg of tryptic peptide mixtures was loaded onto the columns, and separation was done at a flow rate of 2 mL/min by using a linear gradient of 4–50% B for 30 min. A LTQ VELOS (Thermo Electron) equipped with an electrospray interface was connected to the LC setup for eluted peptides detection. Data-dependent MS/MS spectra were ob- tained simultaneously. Each scan cycle consisted of one full MS scan in profile mode followed by five MS/MS scans in centroid mode with the following Dynamic ExclusionTM settings: repeat count 2, repeat duration 30 s, exclusion duration 90 s. Each sample was analyzed in triplicate. MS/MS spectra were automat- ically searched against the non-redundant International Protein Index (IPI) human protein database (version 3.53) using the BioworksBrowser rev. 3.1(Thermo Electron). Immunoprecipitation and Western Blotting Cells were washed with ice-cold PBS and lysed in IP buffer for 1.5 h as described [54]. The lysate was centrifuged and the supernatant was incubated with anti-FLAG M2 affinity gel or anti- HA agarose at 4uC for 8 h. The beads were subsequently washed, and the proteins bound to the beads were eluted. The samples were detected in the subsequent Western procedures with the corresponding antibody. Blots were incubated with IRDye 800CW-conjugated or 700CW-conjugated antibody (Rockland Biosciences) and infrared fluorescence images were obtained with the Odyssey infrared imaging system (Li-Cor Bioscience). Ubiquitination Assay 293 T cells seeded in 60 mm dishes were transfected with different combinations of expression vectors for HA-ubiquitin, GRK5-Flag, and other indicated plasmids. Cells were lysed in ubiqutination buffer (50 mM HEPES, pH 8.0, 250 mM NaCl, 0.5% NP-40, 10% glycerol, 2 mM EDTA, 20 mM NaF, plus 10 mg/ml aprotinin, 10 mg/ml benzamidine, and 0.2 mM PMSF). Cell lysate was immunoprecipitated with anti-Flag M2 affinity gel and analyzed by immunobloting with anti-HA antibody. Role of DDB1 in GRK5 Degradation CHX Treatment For protein decay analysis, 293 T cells were infected by LacZ RNAi or DDB1 RNAi lentivirus for 48 h and then reseeded onto 60 mm culture dish at 76105 cells per dish. After 40 h, cells were exchanged with fresh culture medium and CHX (100 mg/ml) was added at the proper time point for CHX treatment for 0 h, 2 h, 4 h, or 8 h. After CHX treatment, cells were washed with PBS, and total cell lysates were prepared for Western blotting. UV-C Irradiation UV-C (254 nm) irradiation was performed with a UV-C transilluminator (Hoefer UVC 500). The culture medium was replaced with complete medium two hours before the UV-C treatment. Cells were irradiated at 80% confluency with a 20 J/ m2 dose or indicated dose after removal of the medium in the 60 mm petri dish without the lid. After irradiation, cells were post- incubated in their culture medium for the indicated time and were lysed directly in RIPA+ buffer (50 mM Tris.HCl, pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% Deoxycholic Acid, 0.1% SDS, 5 mM EDTA, 10 mM NaF, 10 mM Disodium pyrophosphate, plus 10 mg/ml aprotinin, 10 mg/ml benzamidine, and 0.2 mM PMSF), and equal amount of protein was loaded on gels. Statistical Analysis Data were analyzed using two-way ANOVA for comparison of independent means with pooled estimates of common variances. Supporting Information Table S1 List of proteins associated with GRK5 identi- fied by mass spectrometry in MDA-MB-231 cells. The GRK5 immunocomplex isolated from MDA-MB-231 cells stably expressing GFP or GRK5-Flag were separated on SDS-PAGE and stained with Coomassie Blue. Six pieces of gels (250,400 kDa, 200,250 kDa, 75,200 kDa, 45,60 kDa, 25,30 kDa, 15,20 kDa) were excised and labled as line 1-line 6 as indicate in Fig. 1A. The corresponding control lane was also excised. These gels were analyzed by mass spectrometry. The peptide counts of proteins obtained in GRK5 immunocomplex in MDA-MB-231 cells by mass spectrometry were recorded. Proteins detected in both the control lane and GRK5-immuocomplex lane were excluded from the table. We also provide the peptide counts of proteins identified in HUVEC cells for comparison. The proteins obtained in HUVEC cells but not in MDA-MB-231 cells by mass spectrometry were not listed in the Table S1. (XLSX) Table S2 List of proteins associated with GRK5 identi- fied by mass spectrometry in HUVEC cells. The GRK5 immunocomplex isolated from HUVEC cells transiently trans- fected with GFP or GRK5-Flag were treated as above. The peptide counts of proteins obtained in GRK5 immunocomplex in HUVEC cells by mass spectrometry were recorded. Proteins detected in both the control lane and GRK5-immuocomplex lane were excluded from the table. (XLSX) Table S3 List of protein peptides in GRK5 immuno- complex identified by mass spectrometry in MDA-MB- 231 cells. The peptides of proteins obtained in GRK5 immunocomplex in MDA-MB-231 cells by mass spectrometry were recorded. (XLSX) Table S4 List of protein peptides in GRK5 immuno- complex identified by mass spectrometry in HUVEC PLOS ONE | www.plosone.org 9 August 2012 | Volume 7 | Issue 8 | e43997