Human Family with Sequence Similarity 60 Member A (FAM60A) Protein: a New Subunit of the Sin3 Deacetylase Complex
2012; Elsevier BV; Volume: 11; Issue: 12 Linguagem: Inglês
10.1074/mcp.m112.020255
ISSN1535-9484
AutoresKaren T. Smith, Mihaela E. Sardiu, Skylar Martin‐Brown, Chris Seidel, Arcady Mushegian, Rhonda Egidy, Laurence Florens, Michael P. Washburn, Jerry L. Workman,
Tópico(s)Epigenetics and DNA Methylation
ResumoHere we describe the function of a previously uncharacterized protein, named family with sequence similarity 60 member A (FAM60A) that maps to chromosome 12p11 in humans. We use quantitative proteomics to determine that the main biochemical partners of FAM60A are subunits of the Sin3 deacetylase complex and show that FAM60A resides in active HDAC complexes. In addition, we conduct gene expression pathway analysis and find that FAM60A regulates expression of genes that encode components of the TGF-beta signaling pathway. Moreover, our studies reveal that loss of FAM60A or another component of the Sin3 complex, SDS3, leads to a change in cell morphology and an increase in cell migration. These studies reveal the function of a previously uncharacterized protein and implicate the Sin3 complex in suppressing cell migration. Here we describe the function of a previously uncharacterized protein, named family with sequence similarity 60 member A (FAM60A) that maps to chromosome 12p11 in humans. We use quantitative proteomics to determine that the main biochemical partners of FAM60A are subunits of the Sin3 deacetylase complex and show that FAM60A resides in active HDAC complexes. In addition, we conduct gene expression pathway analysis and find that FAM60A regulates expression of genes that encode components of the TGF-beta signaling pathway. Moreover, our studies reveal that loss of FAM60A or another component of the Sin3 complex, SDS3, leads to a change in cell morphology and an increase in cell migration. These studies reveal the function of a previously uncharacterized protein and implicate the Sin3 complex in suppressing cell migration. Histone deacetylases (HDACs) 1The abbreviations used are:HDAChistone deacetylasesChIPchromatin immunoprecipitationMudPITmultidimensional protein identification technologyAP-MSaffinity purification followed by mass spectrometry. 1The abbreviations used are:HDAChistone deacetylasesChIPchromatin immunoprecipitationMudPITmultidimensional protein identification technologyAP-MSaffinity purification followed by mass spectrometry. catalyze the removal of acetyl groups from proteins. Histones may be the main targets of these enzymes, but they also mediate deacetylation of many nonhistone proteins (1Choudhary C. Kumar C. Gnad F. Nielsen M.L. Rehman M. Walther T.C. Olsen J.V. Mann M. Lysine acetylation targets protein complexes and co-regulates major cellular functions.Science. 2009; 325: 834-840Crossref PubMed Scopus (3152) Google Scholar). There are 11 human histone deacetylases and these fall into four different classes, three of which (Classes I, II, IV) belong to a large superfamily of zinc-dependent deacetylases, and the fourth (Class III, or Sirtuins) are NAD+ dependent (2Witt O. Deubzer H.E. Milde T. Oehme I. HDAC family: What are the cancer relevant targets?.Cancer Letts. 2009; 277: 8-21Crossref PubMed Scopus (817) Google Scholar, 3Yang X.J. Seto E. The Rpd3/Hda1 family of lysine deacetylases: from bacteria and yeast to mice and men.Nat. Rev. Mol. Cell Biol. 2008; 9: 206-218Crossref PubMed Scopus (959) Google Scholar, 4Gregoretti I.V. Lee Y.M. Goodson H.V. Molecular evolution of the histone deacetylase family: functional implications of phylogenetic analysis.J. Mol. Biol. 2004; 338: 17-31Crossref PubMed Scopus (1124) Google Scholar). Of the HDACs, those in Class I (HDACs 1, 2 and 3) are known to reside in large multisubunit complexes that help to specify and diversify HDAC function (3Yang X.J. Seto E. The Rpd3/Hda1 family of lysine deacetylases: from bacteria and yeast to mice and men.Nat. Rev. Mol. Cell Biol. 2008; 9: 206-218Crossref PubMed Scopus (959) Google Scholar). histone deacetylases chromatin immunoprecipitation multidimensional protein identification technology affinity purification followed by mass spectrometry. histone deacetylases chromatin immunoprecipitation multidimensional protein identification technology affinity purification followed by mass spectrometry. HDACs 1 and 2 are found together in at least three distinct protein complexes, Mi-2/Nurd, CoREST and Sin3 (3Yang X.J. Seto E. The Rpd3/Hda1 family of lysine deacetylases: from bacteria and yeast to mice and men.Nat. Rev. Mol. Cell Biol. 2008; 9: 206-218Crossref PubMed Scopus (959) Google Scholar). The Sin3/HDAC complex can affect cell cycle progression through multiple mechanisms (5David G. Grandinetti K.B. Finnerty P.M. Simpson N. Chu G.C. Depinho R.A. 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Sds3 (suppressor of defective silencing 3) is an integral component of the yeast Sin3[middle dot]Rpd3 histone deacetylase complex and is required for histone deacetylase activity.J. Biol. Chem. 2000; 275: 40961-40966Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Other protein subunits are not needed for the integrity of the Sin3 complex and appear to have more peripheral functions. They may mediate interactions with chromatin or sequence specific transcription factors. For example, inhibitor of growth 1 and 2 (ING1 and ING2), tether the complex to specific marks on chromatin (11Ludwig S. Klitzsch A. Baniahmad A. The ING tumor suppressors in cellular senescence and chromatin.Cell Biosci. 2011; 1: 25Crossref PubMed Scopus (17) Google Scholar). Other subunits, such as Sin3, can recruit the complex to different subsets of genes, through interactions with specific transcription factors (12Ayer D.E. Lawrence Q.A. Eisenman R.N. 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Deacetylase inhibitors dissociate the histone-targeting ING2 subunit from the Sin3 complex.Chem. Biol. 2010; 17: 65-74Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). These drugs work through a number of different cellular mechanisms (16Dickinson M. Johnstone R.W. Prince H.M. Histone deacetylase inhibitors: potential targets responsible for their anti-cancer effect.Invest. New Drugs. 2010; 28: S3-20Crossref PubMed Scopus (121) Google Scholar). Biochemically, they bind to the catalytic sites of HDACs and therefore it is assumed that they completely abrogate all of their functions. However, it appears that these drugs may preferentially target some of the noncatalytic subunits of these HDAC complexes (15Smith K.T. Martin-Brown S.A. Florens L. Washburn M.P. Workman J.L. Deacetylase inhibitors dissociate the histone-targeting ING2 subunit from the Sin3 complex.Chem. Biol. 2010; 17: 65-74Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 17Bantscheff M. 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Therefore it is important to understand the complete set of functions encoded by this complex, which subunits mediate these functions and how they are affected by HDAC inhibitors. In this study, we present a systematic quantitative AP-MS analysis of the Sin3/HDAC complex by utilizing multidimensional protein identification technology (MudPIT) and discovered a novel subunit of the Sin3 complex, named FAM60A. This protein lacks homology to other proteins in the human proteome, but is conserved in metazoans. To demonstrate a consistent recovery of FAM60A we examined the complex around six bait proteins. In all, FAM60A was reproducibly detected with high relative abundance values. Reciprocal analysis of FAM60A protein demonstrated a strong association with all selected subunits of the Sin3/HDAC complex. Subsequently, through a series of biochemical approaches and microarray analysis we were able to reveal novel insights into the function of FAM60A protein. Interestingly, we discover that FAM60A represses genes in the TGF-beta signaling pathway. We show that these gene expression changes are linked to FAM60A's role in the Sin3 complex, as SDS3 knockdowns have similar gene expression profiles. Additionally, we find that loss of FAM60A leads to a change in cell morphology and an increase in cell migration in lung cancer and liver cancer cells. These effects are dependent on signaling through the TGF-beta receptors 1 and 2. Importantly, when the SDS3 subunit of the Sin3 complex is abrogated, cells also undergo a change in morphology and an increase in migration. The data suggest that the functions of FAM60A revealed in this study are linked to its occupancy in Sin3. Overall, this work reveals that FAM60A functions in the Sin3 complex as a repressor of TGF-beta signaling and cell migration. 293T cells stably expressing FAM60A-FLAG were produced using the Flp-In system (Invitrogen). Cells were maintained in Dulbecco's modified Eagle's medium (GIBCO) supplemented with 10% fetal bovine serum (FBS). A549 cells were obtained from the American Type Culture Collection (ATCC) and maintained in F-12K media (Cellgro) with 10% FBS. Both cell lines were grown at 37% degrees with 5% CO2 in a humidified atmosphere. Additional cancer cell line lysates were obtained from Aviva Systems Biology or Protein Biotechnologies. Full length cDNA encoding human FAM60A (NCBI Reference Sequence: NM_021238.2) was cloned into pcDNA5-FRT with a single C-terminal FLAG tag. 293T whole cell extracts were made as previously described (Mahrour, 2008). Nuclear extracts were made according to Dignam (23Dignam J.D. Lebovitz R.M. Roeder R.G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei.Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9153) Google Scholar). FLAG purifications from 293T cells were carried out overnight at 4°C using anti-FLAG M2 resin (Sigma). FLAG beads were washed four times for 10 mins each with rotation at 4 °C. Sixty volumes of wash buffer to bead volume were used per wash with the following buffer: 10 mm HEPES pH 7.5, 0.2% Triton X-100, 0.3 m NaCl, 10 mm KCl, and 1.5 mm MgCl2. After washing, proteins were eluted using 3× FLAG peptide (Sigma) diluted to a concentration of 0.2 mg/ml in a buffer containing 50 mm Tris pH 7.5, 0.5% Nonidet P-40, and 0.3 m NaCl. Complexes were then analyzed by MudPIT, silver staining or used in HDAC assays. MudPIT Mass Spectrometry and Database Searching TCA-precipitated protein samples from ING2, SAP30, SAP30L, HDAC1, BRMS1, and BRMS1L Flag-IPs were solubilized in 30 μl of freshly made 0.1 m Tris-HCl, pH 8.5, 8 m urea, 5 mm TCEP (Tris(2-Carboxylethyl)-Phosphine Hydrochloride, Pierce). After 30 min at room temperature, freshly made 0.5 m IAM (Iodoacetamide, Sigma) was added to a final concentration of 10 mm, and the samples were left at room temperature for another 30 min in the dark. Endoproteinase Lys-C (Roche) was first added at an estimated 1:100 (wt/wt) enzyme to protein ratio, for at least 6 h at 37 °C. Urea was then diluted to 2 m with 0.1 m Tris-HCl, pH 8.5, CaCl2 was added to 0.5 mm, and modified trypsin (Promega), 1:100 (wt/wt), was added for over 12 h at 37 °C. All enzymatic digestions were quenched by adding formic acid to 5%. Each trypsin-digested sample was analyzed independently by Multidimensional Protein Identification Technology (MudPIT) as described previously (24Washburn M.P. Wolters D. Yates 3rd, J.R. Large-scale analysis of the yeast proteome by multidimensional protein identification technology.Nat. Biotechnol. 2001; 19: 242-247Crossref PubMed Scopus (4077) Google Scholar, 25Wolters D.A. Washburn M.P. Yates 3rd, J.R. An automated multidimensional protein identification technology for shotgun proteomics.Anal. Chem. 2001; 73: 5683-5690Crossref PubMed Scopus (1566) Google Scholar). Peptide mixtures were pressure-loaded onto a 100 μm fused-silica column pulled to a 5 μm tip using a P 2000 CO2 laser puller (Sutter Instruments). The microcapillary columns were packed first with 8 cm of 5 μm C18 reverse phase (RP) particles (Aqua, Phenomenex), followed by 3 cm of 5 μm strong cation exchange material (Partisphere SCX, Whatman), and by 2 cm of RP particles (26McDonald W.H. Ohi R. Miyamoto D.T. Mitchison T.J. Yates J.R. Comparison of three directly coupled HPLC MS/MS strategies for identification of proteins from complex mixtures: single-dimension LCMS/MS, 2-phase MudPIT, and 3-phase MudPIT.Int. J. Mass Spectrom. 2002; 219: 245-251Crossref Scopus (268) Google Scholar). Loaded microcapillaries were placed in line with LTQ ion trap mass spectrometers (ThermoScientific, San Jose, CA) interfaced with quaternary Agilent 1100 quaternary pumps (Agilent Technologies, Palo Alto, CA). Overflow tubing was used to decrease the flow rate from 0.1 ml/min to about 200–300 nl/min. During the course of a fully automated chromatography, ten 120-min cycles of increasing salt concentrations followed by organic gradients slowly released peptides directly into the mass spectrometer (27Florens L. Washburn M.P. Proteomic analysis by multidimensional protein identification technology.Methods Mol. Biol. 2006; 328: 159-175PubMed Google Scholar). Three different elution buffers were used: 5% acetonitrile, 0.1% formic acid (Buffer A); 80% acetonitrile, 0.1% formic acid (Buffer B); and 0.5 m ammonium acetate, 5% acetonitrile, 0.1% formic acid (Buffer C). The last two chromatography steps consisted of a high salt wash with 100% Buffer C followed by the acetonitrile gradient. The application of a 2.5 kV distal voltage electrosprayed the eluting peptides directly into LTQ linear ion trap mass spectrometers equipped with a nano-LC electrospray ionization source (ThermoFinnigan). Each full MS scan (from 400 to 1600m/z) was followed by five MS/MS events using data-dependent acquisition where the 1st most intense ion was isolated and fragmented by collision-induced dissociation (at 35% collision energy), followed by the 2nd to 5th most intense ions. RAW files were converted to the ms2 format using RAWDistiller v. 1.0, an in-house developed software. The ms2 files were subjected to database searching using SEQUEST (version 27 (rev.9)) with no enzyme specificity considered (28Eng J. McCormack A.L. Yates III, J.R. An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database.J. Am. Mass Spectrom. 1994; 5: 976-989Crossref PubMed Scopus (5420) Google Scholar). The mass tolerance for precursor ions was set at 3 amu, whereas the mass tolerance for fragment ions was 0 amu. MS/MS spectra were searched against a protein database consisting of 29375 nonredundant human proteins (NCBI, 2010-11-22 release) and 160 sequences for usual contaminants (such as keratins, proteolytic enzymes, etc. In addition, to estimate false discovery rates, each nonredundant protein entry was randomized. The resulting "SHUFFLED" sequences were added to the database and searched at the same time as the "forward" sequences. To account for carboxamidomethylation by IAM, +57 Da was added statically to cysteine residues and +16 Da for oxidized methionine residues for all the searches. Results from different runs were compared and merged using CONTRAST (29Tabb D.L. McDonald W.H. Yates 3rd, J.R. DTASelect and Contrast: tools for assembling and comparing protein identifications from shotgun proteomics.J. Proteome Res. 2002; 1: 21-26Crossref PubMed Scopus (1137) Google Scholar). Spectrum/peptide matches were only retained if peptides were at least seven amino acids long and were fully tryptic. The DeltCn had to be at least 0.08, with minimum XCorrs of 1.8 for singly, 2.0 for doubly, and 3.0 for triply charged spectra, and a maximum Sp rank of 10. Finally, combining all runs, proteins had to be detected by at least two such peptides, or one peptide with two independent spectra. Proteins that were subset of others were removed. Quantitation was performed using label-free spectral counting. The number of spectra identified for each protein was used for calculating the distributed normalized spectral abundance factors (dNSAF) (30Zybailov B. Mosley A.L. Sardiu M.E. Coleman M.K. Florens L. Washburn M.P. Statistical analysis of membrane proteome expression changes in Saccharomyces cerevisiae.J. Proteome Res. 2006; 5: 2339-2347Crossref PubMed Scopus (816) Google Scholar). NSAF v7 (an in-house developed software) was used to create the final report on all nonredundant proteins detected across the different runs, estimate false discovery rates (FDR), and calculate their respective distributed Normalized Spectral Abundance Factor (dNSAF) values. Under these criteria the spectral FDRs ranged from 0.00–0.436%, the peptide FDRs ranged from 0.00–0.868% and the protein FDRs ranged from 0.000–2.838% respectively for all pull-downs. Protein-protein interaction networks were created using Cytoscape software (31Shannon P. Markiel A. Ozier O. Baliga N.S. Wang J.T. Ramage D. Amin N. Schwikowski B. Ideker T. Cytoscape: a software environment for integrated models of biomolecular interaction networks.Genome Res. 2003; 13: 2498-2504Crossref PubMed Scopus (25321) Google Scholar). HDAC assays were performed as described previously (15Smith K.T. Martin-Brown S.A. Florens L. Washburn M.P. Workman J.L. Deacetylase inhibitors dissociate the histone-targeting ING2 subunit from the Sin3 complex.Chem. Biol. 2010; 17: 65-74Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Where indicated, trichostatin A was added at a concentration of 1 μm to the assay. Antiserum against human FAM60A was raised in New Zealand White rabbits (Covance), to the following peptide: SSSRFTDSKRYE. The following commercially available antibodies were used: FLAG-HRP (Sigma); Beta-tubulin, Mab380 (Upstate/Millipore); E-cadherin, Ab53033 (Abcam, Cambridge, MA); SDS3, A300–235A (Bethyl Laboratories, Montgomery, TX); ING2, 11560–11561-AP (ProteinTech Group, Inc.). A549 cells were plated at ∼20% confluency the day prior to transfections. Cells were transfected using Dharmafect 1 (Dharmacon) and 50 nm siRNAs (Ambion). Ambion siRNA ID #s are as follows: Control siRNA (AM4637); FAM60A siRNA #1 (s33909); FAM60A siRNA #2 (147804); FAM60A siRNA #3 (s33911); ING2 siRNA (116981); SDS3 siRNA (s34742). Migration assays were carried out in one of two different formats. A. Cells were plated in 6-well plates and transfected 1 day after plating. Scratches were made using a p10 pipette tip when cells reached near confluency. Lines were drawn on the underside of the tissue culture plates to orient scratches and ensure the same region was monitored each day. After scratching, cells were washed, and serum was reduced in the media to 0.5%. B. Alternatively, RADIUS cell migration assays were used (Catalog # CBA-125, Cell Biolabs, Inc.). Cells were transfected 1 day after plating and drugs were added where appropriate 1 day after transfection. Two days (A549s) or 3 days (HepG2s) after transfection, when cells were confluent, the RADIUS gel spot was removed according to the manufacturer's protocol in order to simulate a wound. Media was changed to 0.5% serum and drugs were added during media changes as necessary. Scratches/wounds for all assays were monitored daily. Pictures were taken of the starting scratches/wounds and each day post-scratch, using an Axiovert 200 inverted widefield microscope (Carl Zeiss). Areas were measured by using the "outline" function on the Axiovision program (release 4.7.2). Percent closure was calculated by subtracting the open area over time, from the original starting open area. To minimize variation because of different starting wound sizes, wounds for A549 cells were only considered if they were within ±12% of the average starting scratch/wound size for that experiment. Migration assays were performed on at least three independent transfections for each siRNA. A549 or HepG2 cells were transfected with control siRNAs or siRNAs against FAM60A. Three days after transfection, cells were incubated for 5 hours (A549) or 20 hours (HepG2) with BrdU. BrdU incorporation was detecting using the BrdU and Ki67 cell proliferation kit (Cellomics) according to manufacturer's protocol. Cells were costained with DAPI. Percent incorporation of BrdU in each well was measured by fluorescence analysis using a Celigo Adherent Cell Cytometer (Cyntellect). Data from at least three independent transfections for each siRNA were obtained. RNA was extracted from A549 cells 3 days post-transfection. RNAs from three independent transfections for each siRNA were used. RNA was purified using TRIzol (Invitrogen), and run through an RNeasy clean up column (Qiagen). Concentration and quality of RNA were determined by spectrophotometer and Agilent bioanalyzer analysis (Agilent Technologies, Inc., Palo Alto, CA). For array analysis, labeled mRNA (aRNA) targets were prepared from 1 μg of total RNA using the MessageAmp II-Biotin Enhanced RNA Amplification kit (Applied Biosystems/Ambion, Austin, TX) according to the manufacturer's specifications. Array analysis was performed using Affymetrix GeneChip Human Genome U133 Plus 2.0 Arrays processed with the GeneChip Fluidics Station 450 and scanned with a GeneChip Scanner 3000 7G using standard protocols. Affymetrix CEL files were processed in the R statistical environment and normalized using RMA (32Irizarry R.A. Hobbs B. Collin F. Beazer-Barclay Y.D. Antonellis K.J. Scherf U. Speed T.P. Exploration, normalization, and summaries of high density oligonucleotide array probe level data.Biostatistics. 2003; 4: 249-264Crossref PubMed Scopus (8454) Google Scholar). The linear modeling package Limma (33Smyth G.K. Linear models and empirical bayes methods for assessing differential expression in microarray experiments.Stat. Appl. Genet. Mol. Biol. 2004; 3 (Article 3)Crossref PubMed Scopus (9158) Google Scholar), was used to derive gene expression coefficients and calculate p values. p Values were adjusted for multiple hypothesis testing using the method of Benjamini and Hochberg (34Benjamini Y. Hochber Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing.J. Roy. Stat. Soc. B. 1995; 57: 289-300Google Scholar). The data have been deposited in GEO with accession number: GSE39733, which can be accessed at http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE39733. The secondary structure of FAM60A sequences was predicted with HHPred server (35Söding J. Biegert A. Lupas A.N. The HHpred interactive server for protein homology detection and structure prediction.Nucleic Acids Res. 2005; 33: W244-248Crossref PubMed Scopus (2631) Google Scholar). RNA was purified using TRIzol (Invitrogen), followed by subsequent DNase I digestion and clean up through an RNeasy clean up column (Qiagen). Alternatively, RNA was collected using TRIzol and then purified using the Direct-zol kit (Zymo Research). cDNAs were made using the GeneAmp kit (Applied Biosystems) using random hexamers. Real-time PCR was performed using a BioRad iCycler machine and SYBR green. Cycling conditions are as follows: 3 min at 95 °C, then 41 cycles of: 10 s at 95 °C, 30 s at annealing temperature, and 30 s at 72 °C, then followed by a melt curve. Sequences of primers and annealing temperatures are as follows: FAM60A (Origene): forward 5′-GACTCGTTCAGGAGACATCTGC-3′, reverse 5′-AGTCTTTAGACTGGGTCCAGCC-3′ (annealing at 58 °C); GAPDH: forward 5′-TCCTGCACCACCAACTGCTTAG-3′, reverse 5′-GTAGAGGCAGGGATGATGTTC-3′ (annealing at 58 °C or 60 °C); TGFβR1 (Origene): forward 5′-GACAACGTCAGGTTCTGGCTCA-3′, reverse 5′-CCGCCACTTTCCTCTCCAAACT-3′ (annealing at 60 °C); TGFβ1: forward 5′-TACCTGAACCCGTGTTGCTCTC-3′, reverse 5′-GTTGCTGAGGTATCGCCAGGAA-3′ (annealing at 60 °C); TGFβ2: forward 5′-AAGAAGCGTGCTTTGGATGCGG-3′, reverse 5′-ATGCTCCAGCACAGAAGTTGGC-3′ (annealing at 60 °C); SMAD2 (Origene): forward 5′-GGGTTTTGAAGCCGTCTATCAGC-3′, reverse 5′-CCAACCACTGTAGAGGTCCATTC-3′: ING2: forward 5′-ACATGCAGAGGAACGTGTCTGTG-3′, reverse 5′-ACCAATTCGAGCATTTGTGTAAC-3′. ChIP was performed as described previously (15Smith K.T. Martin-Brown S.A. Florens L. Washburn M.P. Workman J.L. Deacetylase inhibitors dissociate the histone-targeting ING2 subunit from the Sin3 complex.Chem. Biol. 2010; 17: 65-74Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Precipitated DNAs were quantified by real-time PCR. PCR cycling conditions were identical to those used for real-time PCR. Primers used: SMAD2-prom-F: 5′-CCGTAGGCAAAGGGAGGTGGGGA-3′, SMAD2-prom-R: 5′-TTAGGGCGCGAGGCACGCCGGCCGA-3′ (annealing 65 °C); TGFβR1-promoter-F: 5′-CAGCCAATGGACGCGCGTCCT-3′, TGFβR1-promoter-R: 5′-CACCCCAGCAAACCTCGCCT-3′ (annealing at 60 °C). TGFβ1 promoter-F: 5′-TGTTTCCCAGCCTGACTCTCCTT-3′, TGFβ1-promoter-R: 5′-ACCAAAGCGGGTGATCCAGAT-3′. To expand the network of the Sin3/HDAC complex, we selected and purified six of the known core subunits (BRMS1, BRMS1L, ING2, SAP30, SAP30L and HDAC1) of the Sin3/HDAC complex. We performed at least three replicates for each of the baits to ensure good reproducibility of the data. To control for nonspecific proteins, we additionally performed ten purifications from a control 293T cell line. A total of 29 purifications (including replicates) were carried out, which led to a total of 3345 identified proteins (Supplemental Tables S1 and S2). Quantitation was performed using label-free spectral counting. The number of spectra identified for each protein was used to calculate the normalized abundance factors. NSAF7 software developed in house (36Mosley A.L. Florens L. Wen Z. Washburn M.P. A label free quantitative proteomic analysis of the Saccharomyces cerevisiae nucleus.J. Proteomics. 2009; 72: 110-120Crossref PubMed Scopus (53) Google Scholar, 37Zhang Y. Wen Z. Washburn M.P. Florens L. Refinements to label free proteome quantitation: how to deal with peptides shared by multiple proteins.Anal. Chem. 2010; 82: 2272-2281Crossref PubMe
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