BioID-based Identification of Skp Cullin F-box (SCF)β-TrCP1/2 E3 Ligase Substrates*
2015; Elsevier BV; Volume: 14; Issue: 7 Linguagem: Inglês
10.1074/mcp.m114.045658
ISSN1535-9484
AutoresÉtienne Coyaud, Monika Mis, Estelle Laurent, Wade H. Dunham, Amber L. Couzens, Mélanie Robitaille, Anne‐Claude Gingras, Stéphane Angers, Brian Raught,
Tópico(s)Protein Degradation and Inhibitors
ResumoThe identification of ubiquitin E3 ligase substrates has been challenging, due in part to low-affinity, transient interactions, the rapid degradation of targets and the inability to identify proteins from poorly soluble cellular compartments. SCFβ-TrCP1 and SCFβ-TrCP2 are well-studied ubiquitin E3 ligases that target substrates for proteasomal degradation, and play important roles in Wnt, Hippo, and NFκB signaling. Combining 26S proteasome inhibitor (MG132) treatment with proximity-dependent biotin labeling (BioID) and semiquantitative mass spectrometry, here we identify SCFβ-TrCP1/2 interacting partners. Based on their enrichment in the presence of MG132, our data identify over 50 new putative SCFβ-TrCP1/2 substrates. We validate 12 of these new substrates and reveal previously unsuspected roles for β-TrCP in the maintenance of nuclear membrane integrity, processing (P)-body turnover and translational control. Together, our data suggest that β-TrCP is an important hub in the cellular stress response. The technique presented here represents a complementary approach to more standard IP-MS methods and should be broadly applicable for the identification of substrates for many ubiquitin E3 ligases. The identification of ubiquitin E3 ligase substrates has been challenging, due in part to low-affinity, transient interactions, the rapid degradation of targets and the inability to identify proteins from poorly soluble cellular compartments. SCFβ-TrCP1 and SCFβ-TrCP2 are well-studied ubiquitin E3 ligases that target substrates for proteasomal degradation, and play important roles in Wnt, Hippo, and NFκB signaling. Combining 26S proteasome inhibitor (MG132) treatment with proximity-dependent biotin labeling (BioID) and semiquantitative mass spectrometry, here we identify SCFβ-TrCP1/2 interacting partners. Based on their enrichment in the presence of MG132, our data identify over 50 new putative SCFβ-TrCP1/2 substrates. We validate 12 of these new substrates and reveal previously unsuspected roles for β-TrCP in the maintenance of nuclear membrane integrity, processing (P)-body turnover and translational control. Together, our data suggest that β-TrCP is an important hub in the cellular stress response. The technique presented here represents a complementary approach to more standard IP-MS methods and should be broadly applicable for the identification of substrates for many ubiquitin E3 ligases. More than 600 putative ubiquitin E3 ligases are encoded in the human genome (1.Varshavsky A. The ubiquitin system, an immense realm.Annu. Rev. Biochem. 2012; 81: 167-176Crossref PubMed Scopus (218) Google Scholar, 2.Hershko A. Ciechanover A. The ubiquitin system.Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6880) Google Scholar). Although a number of these proteins are known to play critical roles in human health (1.Varshavsky A. The ubiquitin system, an immense realm.Annu. Rev. Biochem. 2012; 81: 167-176Crossref PubMed Scopus (218) Google Scholar, 2.Hershko A. Ciechanover A. The ubiquitin system.Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6880) Google Scholar, 3.Scheffner M. Staub O. HECT E3s and human disease.BMC Biochem. 2007; 8: S6Crossref PubMed Scopus (78) Google Scholar, 4.Jiang Y.H. Beaudet A.L. Human disorders of ubiquitination and proteasomal degradation.Curr. Opin. Pediar. 2004; 16: 419-426Crossref PubMed Scopus (72) Google Scholar), the specific biological functions–and substrates–of most E3s remain poorly characterized. The identification of E3 substrates has been difficult in part because: (1) ligase - substrate interactions are often of low affinity (generally in the high nm to microMolar (μM) range) and/or of a transient nature; (2) many substrates are subjected to rapid proteasomal degradation and are therefore not available for detection; (3) the human ubiquitome is extremely complex, and; (4) many substrate proteins are localized to poorly soluble cellular compartments, making their isolation and identification by standard immunoprecipitation (IP)-based techniques extremely challenging (1.Varshavsky A. The ubiquitin system, an immense realm.Annu. Rev. Biochem. 2012; 81: 167-176Crossref PubMed Scopus (218) Google Scholar, 2.Hershko A. Ciechanover A. The ubiquitin system.Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6880) Google Scholar, 3.Scheffner M. Staub O. HECT E3s and human disease.BMC Biochem. 2007; 8: S6Crossref PubMed Scopus (78) Google Scholar, 4.Jiang Y.H. Beaudet A.L. Human disorders of ubiquitination and proteasomal degradation.Curr. Opin. Pediar. 2004; 16: 419-426Crossref PubMed Scopus (72) Google Scholar). Methods such as protein chip (5.Persaud A. Rotin D. Use of proteome arrays to globally identify substrates for E3 ubiquitin ligases.Methods Mol. Biol. 2011; 759: 215-224Crossref PubMed Scopus (6) Google Scholar) and yeast two-hybrid screening (6.Rual J.F. Venkatesan K. Hao T. Hirozane-Kishikawa T. Dricot A. Li N. Berriz G.F. Gibbons F.D. Dreze M. Ayivi-Guedehoussou N. Klitgord N. Simon C. Boxem M. Milstein S. Rosenberg J. Goldberg D.S. Zhang L.V. Wong S.L. Franklin G. Li S. Albala J.S. Lim J. Fraughton C. Llamosas E. Cevik S. Bex C. Lamesch P. Sikorski R.S. Vandenhaute J. Zoghbi H.Y. Smolyar A. Bosak S. Sequerra R. Doucette-Stamm L. Cusick M.E. Hill D.E. Roth F.P. Vidal M. Towards a proteome-scale map of the human protein-protein interaction network.Nature. 2005; 437: 1173-1178Crossref PubMed Scopus (2284) Google Scholar) have been used to identify a limited number of E3-substrate interactions. However, these methods are not conducted in live mammalian cells, and may not be generally applicable for the identification of substrates of the hundreds of unique multi-protein E3 complexes (e.g. SCF, APC, VHL, etc.) or the identification of E3-substrate interactions that are dependent on specific types of post-translational modifications. Several putative inhibitors of apoptosis substrates were identified using the recently described NEDDylator technique (7.Zhuang M. Guan S. Wang H. Burlingame A.L. Wells J.A. Substrates of IAP ubiquitin ligases identified with a designed orthogonal E3 ligase, the NEDDylator.Mol. Cell. 2013; 49: 273-282Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar), in which the E2 protein UBC12 (UBE2M) is fused to the E3 ligase of interest, allowing for the conjugation of the ubiquitin-like protein NEDD8 to substrates. Global protein stability profiling (8.Yen H.C. Elledge S.J. Identification of SCF ubiquitin ligase substrates by global protein stability profiling.Science. 2008; 322: 923-929Crossref PubMed Scopus (151) Google Scholar) and quantitative mass spectrometry methods (9.Hor S. Ziv T. Admon A. Lehner P.J. Stable isotope labeling by amino acids in cell culture and differential plasma membrane proteome quantitation identify new substrates for the MARCH9 transmembrane E3 ligase.Mol. Cell. Proteomics. 2009; 8: 1959-1971Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) have also been used successfully to identify E3 targets. However, because of their cost and/or complexity, and the challenges posed by the extremely large size of the human ubiquitome, these methods have not been widely adopted to date. Proximity-based biotinylation, or BioID 1The abbreviations used are:BioIDproximity-based biotinylationCHXcyclohexamideEDMDEmery-Dreifuss Muscular Dystrophy. 1The abbreviations used are:BioIDproximity-based biotinylationCHXcyclohexamideEDMDEmery-Dreifuss Muscular Dystrophy. (10.Roux K.J. Kim D.I. Raida M. Burke B. A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells.J. Cell Biol. 2012; 196: 801-810Crossref PubMed Scopus (1234) Google Scholar), is a new method developed for the characterization of protein-protein interactions in living cells. Briefly, a protein of interest is fused in-frame with an E. coli biotin conjugating enzyme mutant (BirA R118G, or BirA*). The BirA* moiety can efficiently activate biotin, but exhibits a reduced affinity for the activated molecule (11.Kwon K. Streaker E.D. Beckett D. Binding specificity and the ligand dissociation process in the E. coli biotin holoenzyme synthetase.Protein Sci. 2002; 11: 558-570Crossref PubMed Scopus (28) Google Scholar); biotinoyl-AMP thus simply diffuses away from BirA* and reacts with nearby amine groups - including those present on lysine residues in neighboring polypeptides. Following cell lysis, biotinylated proteins can be affinity purified using streptavidin and identified using mass spectrometry (Fig. 1A). Since interactors are covalently modified with biotin, robust lysis conditions can be used to solubilize polypeptides localized to poorly soluble cellular compartments (10.Roux K.J. Kim D.I. Raida M. Burke B. A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells.J. 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We reasoned that BioID may be exploited to capture ubiquitin E3 ligase substrates and tested this notion here. proximity-based biotinylation cyclohexamide Emery-Dreifuss Muscular Dystrophy. proximity-based biotinylation cyclohexamide Emery-Dreifuss Muscular Dystrophy. The human beta transducin repeat-containing polypeptides β-TrCP1 (FBXW1) and β-TrCP2 (FBXW11) are evolutionarily conserved paralogous F-box proteins sharing >80% amino acid sequence identity and >90% homology, and act as substrate recognition components of SCF (Skp1-Cullin-F-box) complexes (17.Willems A.R. Schwab M. Tyers M. A hitchhiker's guide to the cullin ubiquitin ligases: SCF and its kin.Biochim. Biophys. Acta. 2004; 1695: 133-170Crossref PubMed Scopus (379) Google Scholar). A number of β-TrCP substrates have been well documented, implicating these ligases in numerous biological functions, including regulation of the NFκB, Hippo, Wnt and Hedgehog signaling pathways (18.Kanarek N. Ben-Neriah Y. 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Here we demonstrate that BioID performed on cells treated with the proteasome inhibitor MG132 can recover many of the previously characterized substrates and stable interactors of β-TrCP1/2. Using semi-quantitative mass spectrometry, we identify and validate a number of new substrates, linking these well-studied E3 ligases to several new biological functions. The method used here is simple, scalable, and should be broadly applicable for the identification of substrates for many other E3s. All PCR reactions were performed using Q5 DNA polymerase (NEB) according to manufacturer's instructions. Primers, destination plasmids, cloning sites and DNA templates are listed in supplemental Table S4. PCR products were digested with AscI and NotI (NEB) and ligated into the destination plasmid using T4 DNA ligase (NEB) following manufacturer's instructions. The PPP1R15B S459–466A phosphodegron mutant was generated by PCR-driven overlap extension (23.Heckman K.L. Pease L.R. Gene splicing and mutagenesis by PCR-driven overlap extension.Nat. Protoc. 2007; 2: 924-932Crossref PubMed Scopus (857) Google Scholar) using WT PPP1R15B external primers and the overlapping internal primers listed in supplemental Table S4. BioID (10.Roux K.J. Kim D.I. Raida M. Burke B. A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells.J. Cell Biol. 2012; 196: 801-810Crossref PubMed Scopus (1234) Google Scholar) was carried out essentially as we described previously (12.Comartin D. Gupta G.D. Fussner E. Coyaud E. Hasegan M. Archinti M. Cheung S.W. Pinchev D. Lawo S. Raught B. Bazett-Jones D.P. Luders J. Pelletier L. CEP120 and SPICE1 cooperate with CPAP in centriole elongation.Curr. Biol. 2013; 23: 1360-1366Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). In brief, full length human β-TrCP1 (BC027994) and β-TrCP2 (BC026213) coding sequences were amplified by PCR and cloned into our pcDNA5 FRT/TO FLAGBirA* expression vector (see primers in supplemental Table S4). Using the Flp-In system (Invitrogen), 293 T-REx Flp-In cells stably expressing FLAGBirA*-β-TrCP1 or FLAGBirA*-β-TrCP2 were generated. After selection (DMEM + 10% FBS + 200 μg/ml Hygromycin B), 10 × 150 cm2 plates of sub-confluent (60%) cells were incubated for 24 h in complete media supplemented with 1 μg/ml tetracycline (Sigma) and 50 μm biotin (BioShop, Burlington, ON, Canada). Five plates were treated with 5 μm MG132 (calpain inhibitor IV, Z-Leu-Leu-Leu-CHO; American Peptide Company, Sunnyvale, CA). Cells were collected and pelleted (2000 rpm, 3 min), the pellet was washed twice with PBS, and dried pellets were snap frozen. Pellets were lysed in 10 ml of modified RIPA lysis buffer (50 mm Tris-HCl pH 7.5, 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1% Triton X-100, 0.1% SDS, 1:500 protease inhibitor mixture (Sigma-Aldrich, Saint-Louis, MO), 250U Turbonuclease (Accelagen, San Diego, CA)) at 4°C for 1 h, then sonicated (30 s at 35% power, Sonic Dismembrator 500; Fisher Scientific) to disrupt visible aggregates. The lysate was centrifuged at 16000 rpm (35000 × g) for 30 min. Clarified supernatants were incubated with 30 μl packed, pre-equilibrated streptavidin-Sepharose beads (GE) at 4 °C for 3 h. Beads were collected by centrifugation (2000 rpm, 2 min), washed six times with 50 mm ammonium bicarbonate pH 8.3, and treated with TPCK-trypsin (Promega, Madison, WI, 16 h at 37 °C). The supernatant containing the tryptic peptides was collected and lyophilized. Peptides were resuspended in 0.1% formic acid and 1/6th of the sample was analyzed per MS run. For FLAG pulldowns, 10 × 150 cm2 dishes of FLAGBirA*-β-TrCP1 or FLAGBirA*-β-TrCP2 293 T-REx cells were treated with tetracycline and biotin as above (5 plates were treated with 5 μm MG132), scraped into PBS, pooled, washed twice in 25 ml PBS and collected by centrifugation at 1000 × g for 5 min at 4 °C. Cell pellets were stored at −80 °C until lysis. The cell pellet was weighed and 1:4 pellet weight/lysis buffer (by volume) was added. Lysis buffer consisted of 50 mm HEPES-NaOH (pH 8.0), 100 mm KCl, 2 mm EDTA, 0.1% Nonidet P-40, 10% glycerol, 1 mm PMSF, 1 mm DTT and 1:500 protease inhibitor mixture (Sigma-Aldrich, St. Louis, MO). Upon resuspension, cells were incubated on ice for 10 min, subjected to one additional freeze-thaw cycle, then centrifuged at 27000 × g for 20 min at 4 °C. Supernatant was transferred to a fresh 15 ml conical tube and 250U Turbonuclease (Accelagen) plus 30 μl packed, pre-equilibrated FLAG-M2 agarose beads (Sigma-Aldrich) were added. The mixture was incubated for 2 h at 4 °C with end-over-end rotation. Beads were pelleted by centrifugation at 1000 rpm (100 × g) for 1 min and transferred with 1 ml of lysis buffer to a fresh centrifuge tube. Beads were washed once with 1 ml lysis buffer and twice with 1 ml ammonium bicarbonate (ammbic) rinsing buffer (50 mm ammbic pH 8.0, 75 mm KCl). Elution was performed by incubating the beads with 150 μl of 125 mm ammonium hydroxide (pH >11). The elution step was repeated twice more, and the combined eluate centrifuged at 15000 × g for 10 min, transferred to a fresh centrifuge tube and lyophilized. Following overnight trypsin digestion (as above), peptides were resuspended in 0.1% formic acid and 1/6th of the sample was analyzed per MS run. Liquid chromatography (LC) analytical columns (75 μm inner diameter) and precolumns (150 μm ID) were made in-house from fused silica capillary tubing from InnovaQuartz (Phoenix, AZ) and packed with 100Å C18-coated silica particles (Magic, Michrom Bioresources, Auburn, CA). LC-MS/MS was conducted using a 120 min reversed-phase buffer gradient running at 250 nl/min (column heated to 40 °C) on a Proxeon EASY-nLC pump in-line with a hybrid LTQ-Orbitrap velos mass spectrometer (Thermo Fisher Scientific). A parent ion scan was performed in the Orbitrap, using a resolving power of 60000. Simultaneously, up to the twenty most intense peaks were selected for MS/MS (minimum ion count of 1000 for activation) using standard CID fragmentation. Fragment ions were detected in the LTQ. Dynamic exclusion was activated such that MS/MS of the same m/z (within a 10 ppm window, exclusion list size 500) detected three times within 45 s were excluded from analysis for 30 s. For protein identification, .raw files were converted to the .mzXML format using Proteowizard (24.Kessner D. Chambers M. Burke R. Agus D. Mallick P. ProteoWizard: open source software for rapid proteomics tools development.Bioinformatics. 2008; 24: 2534-2536Crossref PubMed Scopus (1218) Google Scholar), then searched using X!Tandem (25.Craig R. Beavis R.C. TANDEM: matching proteins with tandem mass spectra.Bioinformatics. 2004; 20: 1466-1467Crossref PubMed Scopus (1987) Google Scholar) against Human RefSeq Version 45 (containing 36113 entries). Search parameters specified a parent MS tolerance of 15 ppm and an MS/MS fragment ion tolerance of 0.4 Da, with up to two missed cleavages allowed for trypsin. Oxidation of methionine was allowed as a variable modification. Data were analyzed using the trans-proteomic pipeline (26.Pedrioli P.G. Trans-proteomic pipeline: a pipeline for proteomic analysis.Methods Mol. Biol. 2010; 604: 213-238Crossref PubMed Scopus (115) Google Scholar) via the ProHits 2.0.0 software suite (27.Liu G. Zhang J. Larsen B. Stark C. Breitkreutz A. Lin Z.Y. Breitkreutz B.J. Ding Y. Colwill K. Pasculescu A. Pawson T. Wrana J.L. Nesvizhskii A.I. Raught B. Tyers M. Gingras A.C. ProHits: integrated software for mass spectrometry-based interaction proteomics.Nat. Biotechnol. 2010; 28: 1015-1017Crossref PubMed Scopus (151) Google Scholar). Proteins identified with a ProteinProphet cut-off of 0.85 (corresponding to ≤1% FDR) were analyzed with SAINT Express v. 3.3 (28.Choi H. Larsen B. Lin Z.Y. Breitkreutz A. Mellacheruvu D. Fermin D. Qin Z.S. Tyers M. Gingras A.C. Nesvizhskii A.I. SAINT: probabilistic scoring of affinity purification-mass spectrometry data.Nat. Methods. 2011; 8: 70-73Crossref PubMed Scopus (481) Google Scholar, 29.Teo G. Liu G. Zhang J. Nesvizhskii A.I. Gingras A.C. Choi H. SAINTexpress: improvements and additional features in Significance Analysis of INTeractome software.J. Proteomics. 2014; 100: 37-43Crossref PubMed Scopus (291) Google Scholar). For BioID, 24 control runs were used for comparative purposes; eight runs of a BioID analysis conducted on cells expressing the FlagBirA* tag only, eight runs of BioID conducted on untransfected 293 T-REx cells, and eight runs from a BioID analysis conducted on an unrelated bait protein. In each case, four runs were conducted on untreated cells and four runs were conducted on cells treated with MG132, as above. The 24 controls were collapsed to the highest four spectral counts for each hit. Similarly, for Flag IP-MS analysis, 24 control runs were conducted; FLAG IP-MS analyses of three unrelated FLAGBirA*-tagged proteins expressed in 293 T-REx cells, in the presence and absence of MG132. The 24 control runs were collapsed to the highest four spectral counts for each hit. All data are publically available and have been uploaded to the MassIVE archive (massive.ucsd.edu) ID: MSV000079032. Annotated spectra are available for inspection via the MS-Viewer tool (30.Baker P.R. Chalkley R.J. MS-viewer: a web-based spectral viewer for proteomics results.Mol. Cell. Proteomics. 2014; 13: 1392-1396Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar) of Protein Prospector (prospector2.ucsf.edu; see supplemental Table S1 for all associated file keys). Based on four independent MS runs (two technical replicates and two biological replicates for each bait, in the presence and absence of MG132), bona fide interactors were defined as high confidence protein identifications (ProteinProphet p value >0.85) displaying a SAINT score ≥0.75. Histones were removed manually. Fold-change was calculated as: log2 (1+ spectral count sum in the presence of MG132)/(1+ spectral count sum in the absence of MG132). Statistical significance of the difference between the spectral counts detected in samples prepared from cells cultured with and without MG132 was calculated using Student's t test. Hits considered as putative substrates display a log2 fold change ≥ 1 (Table I) and p value ≤ 0.05. Gene ontology (GO) category assignments detailed in supplemental Table S3.Table Iβ-TrCP BioID and FLAG IP-MS hits displaying a "substrate profile." FLAGBirA*-β-TrCP1 and FLAGBirA*-β-TrCP2 BioID and FLAG IP-MS results. Data are presented as spectral counts detected for each prey protein, as indicated. Two technical replicates were performed on each of two unique biological replicates for each bait protein (for a total of four MS analyses), in both the presence and absence of MG132 (5uM, 24 hrs). ProteinProphet confidence score p ≥ 0.85, SAINT score ≥0.75. For control runs, only the highest four spectral counts (out of 24 runs) are shown. *log2 ratio of total spectral counts detected for each of the indicated polypeptides in the presence vs. the absence of MG132. One spectral count was added to all totals for this analysis, to avoid division by 0. All ratios shown were statistically significant (p < 0.05) as determined by student's t-test (blank values not significant and/or not identified as a high confidence interactor with the corresponding technique and bait protein). β-TrCP1 substrate candidates indicated in light blue, β-TrCP2 substrate candidates indicated in green. Previously reported β-TrCP1/2 interactor gene names are highlighted in bold. Proteins demonstrated here to be stabilized following β-TrCP knockdown (Fig. 2 and supplementary Fig. S2) are boxed in dark blue, with white font At day 0, 5 × 105 293 T-REx Flp-In cells were transfected with 20 nm siRNA Control (siCONTROL non-targeting siRNA #1 Thermo Scientific); 10 nm siRNA β-TrCP1: CGGAAGAGUUUUUCGACUAtt (predesigned Invitrogen ID:s17110) + 10 nm siRNA β-TrCP2: GGUUGUUAGUGGAUCAUCAtt (predesigned Invitrogen ID:s23485); or 20 nm siRNA β-TrCP1/2: GUGGAAUUUGUGGAACAUCtt (31.Tsuchiya Y. Morita T. Kim M. Iemura S. Natsume T. Yamamoto M. Kobayashi A. Dual regulation of the transcriptional activity of Nrf1 by beta-TrCP- and Hrd1-dependent degradation mechanisms.Mol. Cell. Biol. 2011; 31: 4500-4512Crossref PubMed Scopus (75) Google Scholar) using Lipofectamine RNAiMAX (Invitrogen), according to manufacturer's instructions. On day one, transfection media was removed and replaced by fresh media, and cells were subjected to a second transfection with epitope-tagged (3xHA, FLAG or FLAGBirA*) substrate candidates in pcDNA3 or pcDNA5 using PolyJet transfection reagent (Signagen, Rockville, MD) according to manufacturer's instructions. On day 2, cells were trypsinized and 105 cells/well were distributed to a 24-well plate. On day 3, CHX (100 μg/ml) was added for 0–8 h. Upon collection, media was removed and 80 μl of 95 °C SDS lysis buffer (50 mm TrisHCl pH 6.8, 2% SDS, 20% glycerol, 50 mm DTT, bromphenol blue) was added to each well. Cells in lysis buffer were scraped and transferred to an Eppendorf tube, incubated 5 min at 95 °C, and briefly sonicated. Twenty microliters of each sample was loaded on a 4–12% Criterion™ XT Bis-Tris Gel (Bio-Rad, Hercules, MD). Proteins were transferred to nitrocellulose (BioTrace NT (Pall, Mississauga, ON, Canada) CA27376–991) and blocked 1 h in PBS 0.01% Tween + 5% skim milk or 5% BSA. Antibodies were diluted in blocking agent and used as follows: HA.11 Clone 16B2 (Covance, Princeton, NJ) 1:5000 16 h 4 °C; FLAG M2 (Sigma) 1:2000 16 h 4 °C; anti-β-actin (C4; Santa-Cruz, Dallas, TX) 1:2000 16 h 4 °C; mAb414 (Covance) 1:5000 16 h 4 °C, Streptactin-HRP 1:5000 1 h RT. Membranes were washed with PBS 0.01% Tween, and incubated 1 h in PBS 0.01% Tween with 1:10000 Goat anti-Mouse IgG (H + L)-HRP Conjugate (BioRad #1706516). Membranes were washed four times with PBS 0.01% Tween and revealed using Clarity Western ECL substrate (Bio-Rad, Hercules, MD) enhanced chemiluminescence reagent. Band intensities were quantified using ImageJ software, using arbitrary pixel intensity units. Each 3xHA or FLAG signal was normalized to the corresponding anti-β-actin signal for the same lane. Each lane is then presented as a fraction of the band intensity at time zero for that treatment. Untransfected HeLa cells or HeLa stable cell lines expressing FLAG-SUN2 were grown on poly-l-Lysine (Sigma) coated coverslips, fixed with 4% formaldehyde for 15 min, and washed in PBS with 0.1% Triton X-100. Cells were blocked in 5% BSA in PBS for 30 min before incubating in PBS with 1:1000 anti-FLAG M2 (Sigma), anti-Lamin B (Abcam) or anti-SUN2 (Sigma) primary antibodies for 1h at RT. Anti-mouse Alexa 488 and Streptavidin-Alexa594 (Life Technologies) were used at 1:1000 and 1:5000, respectively, and incubated at RT for 1 h. After removing the solution, cells were incubated with 1 μg/ml of 4′,6-diamidino-2-phenylindole (DAPI) in PBS for 5 min. After washing with PBS 3 × 5 min, coverslips were mounted with ProLong Gold Antifade (Thermo Fischer Scientific). Cells were imaged using a PlanApo 60X oil lens, NA 1.40 on an Olympus FV1000 confocal microscope (zoom factor between 3 and 5; Olympus America, Melville, NY). Images were processed using the Volocity Viewer v.6 and assembled using Adobe Illustrator CS5 (Adobe System Inc., San Jose, CA). GFP and mCherry constructs were generated by Gateway cloning into an N- or C-terminal pDEST pcDNA5/FRT/TO-eGFP, or an N-terminal pDEST pcDNA5/FRT/TO-mCherry vector. Clone accession numbers are shown in supplemental Table S4. Stable Flp-In HeLa T-REx cell line pools were generated as described in (32.Kean M.J. Couzens A.L. Gingras A.C. Mass spectrometry approaches to study mammalian kinase and phosphatase associated proteins.Methods. 2012; 57: 400-408Crossref PubMed Scopus (51) Google Scholar). The PATL1, DCP1A, EIF4A1, PABPC1 and G3BP1 genes were cloned from the following clone accession number templates: AM392959, DQ893260, DQ893044, DQ893508, and DQ893058. For live-cell studies, cultured HeLa cells transiently transfected with pcDNA3 Cherry-SUN2 or stably transfected with the GFP or mCherry-tagged P-body or stress granule markers were transfected with siRNA control or siRNA β-TrCP1/2 in 24-well plates, incubated 48 h, then seeded into Lab-Tek chambers (Thermo Fisher) and treated for 24 h with 1 μg/ml tetracycline to induce marker expression. HeL
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