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1.
J Proteome Res ; 23(8): 3704-3715, 2024 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-38943634

RESUMO

Proteome coverage and accurate protein quantification are both important for evaluating biological systems; however, compromises between quantification, coverage, and mass spectrometry (MS) resources are often necessary. Consequently, experimental parameters that impact coverage and quantification must be adjusted, depending on experimental goals. Among these parameters is offline prefractionation, which is utilized in MS-based proteomics to decrease sample complexity resulting in higher overall proteome coverage upon MS analysis. Prefractionation leads to increases in required MS analysis time, although this is often mitigated by isobaric labeling using tandem-mass tags (TMT), which allow samples to be multiplexed. Here we evaluate common prefractionation schemes, TMT variants, and MS acquisition methods and their impact on protein quantification and coverage. Furthermore, we provide recommendations for experimental design depending on the experimental goals.


Assuntos
Proteoma , Proteômica , Espectrometria de Massas em Tandem , Proteômica/métodos , Proteômica/normas , Espectrometria de Massas em Tandem/métodos , Proteoma/análise , Humanos , Fracionamento Químico/métodos , Coloração e Rotulagem/métodos
2.
Nucleic Acids Res ; 45(11): 6698-6716, 2017 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-28334900

RESUMO

CDK12 (cyclin-dependent kinase 12) is a regulatory kinase with evolutionarily conserved roles in modulating transcription elongation. Recent tumor genome studies of breast and ovarian cancers highlighted recurrent CDK12 mutations, which have been shown to disrupt DNA repair in cell-based assays. In breast cancers, CDK12 is also frequently co-amplified with the HER2 (ERBB2) oncogene. The mechanisms underlying functions of CDK12 in general and in cancer remain poorly defined. Based on global analysis of mRNA transcripts in normal and breast cancer cell lines with and without CDK12 amplification, we demonstrate that CDK12 primarily regulates alternative last exon (ALE) splicing, a specialized subtype of alternative mRNA splicing, that is both gene- and cell type-specific. These are unusual properties for spliceosome regulatory factors, which typically regulate multiple forms of alternative splicing in a global manner. In breast cancer cells, regulation by CDK12 modulates ALE splicing of the DNA damage response activator ATM and a DNAJB6 isoform that influences cell invasion and tumorigenesis in xenografts. We found that there is a direct correlation between CDK12 levels, DNAJB6 isoform levels and the migration capacity and invasiveness of breast tumor cells. This suggests that CDK12 gene amplification can contribute to the pathogenesis of the cancer.


Assuntos
Processamento Alternativo , Neoplasias da Mama/genética , Quinases Ciclina-Dependentes/fisiologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular , Reparo do DNA , Éxons , Feminino , Amplificação de Genes , Regulação Neoplásica da Expressão Gênica , Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP40/metabolismo , Humanos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Invasividade Neoplásica , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Poliadenilação , Mapas de Interação de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
3.
Nature ; 486(7403): 395-9, 2012 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-22495314

RESUMO

Primary triple-negative breast cancers (TNBCs), a tumour type defined by lack of oestrogen receptor, progesterone receptor and ERBB2 gene amplification, represent approximately 16% of all breast cancers. Here we show in 104 TNBC cases that at the time of diagnosis these cancers exhibit a wide and continuous spectrum of genomic evolution, with some having only a handful of coding somatic aberrations in a few pathways, whereas others contain hundreds of coding somatic mutations. High-throughput RNA sequencing (RNA-seq) revealed that only approximately 36% of mutations are expressed. Using deep re-sequencing measurements of allelic abundance for 2,414 somatic mutations, we determine for the first time-to our knowledge-in an epithelial tumour subtype, the relative abundance of clonal frequencies among cases representative of the population. We show that TNBCs vary widely in their clonal frequencies at the time of diagnosis, with the basal subtype of TNBC showing more variation than non-basal TNBC. Although p53 (also known as TP53), PIK3CA and PTEN somatic mutations seem to be clonally dominant compared to other genes, in some tumours their clonal frequencies are incompatible with founder status. Mutations in cytoskeletal, cell shape and motility proteins occurred at lower clonal frequencies, suggesting that they occurred later during tumour progression. Taken together, our results show that understanding the biology and therapeutic responses of patients with TNBC will require the determination of individual tumour clonal genotypes.


Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Evolução Molecular , Mutação/genética , Alelos , Neoplasias da Mama/diagnóstico , Células Clonais/metabolismo , Células Clonais/patologia , Variações do Número de Cópias de DNA/genética , Análise Mutacional de DNA , Progressão da Doença , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/genética , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação INDEL/genética , Mutação Puntual/genética , Medicina de Precisão , Reprodutibilidade dos Testes , Análise de Sequência de RNA
4.
Blood ; 123(25): 3914-24, 2014 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-24802772

RESUMO

The histone methyltransferase EZH2 is frequently mutated in germinal center-derived diffuse large B-cell lymphoma and follicular lymphoma. To further characterize these EZH2 mutations in lymphomagenesis, we generated a mouse line where EZH2(Y641F) is expressed from a lymphocyte-specific promoter. Spleen cells isolated from the transgenic mice displayed a global increase in trimethylated H3K27, but the mice did not show an increased tendency to develop lymphoma. As EZH2 mutations often coincide with other mutations in lymphoma, we combined the expression of EZH2(Y641F) by crossing these transgenic mice with Eµ-Myc transgenic mice. We observed a dramatic acceleration of lymphoma development in this combination model of Myc and EZH2(Y641F). The lymphomas show histologic features of high-grade disease with a shift toward a more mature B-cell phenotype, increased cycling and gene expression, and epigenetic changes involving important pathways in B-cell regulation and function. Furthermore, they initiate disease in secondary recipients. In summary, EZH2(Y641F) can collaborate with Myc to accelerate lymphomagenesis demonstrating a cooperative role of EZH2 mutations in oncogenesis. This murine lymphoma model provides a new tool to study global changes in the epigenome caused by this frequent mutation and a promising model system for testing novel treatments.


Assuntos
Transformação Celular Neoplásica/genética , Linfoma/genética , Mutação , Complexo Repressor Polycomb 2/genética , Proteínas Proto-Oncogênicas c-myc/genética , Animais , Linfócitos B/metabolismo , Linfócitos B/patologia , Western Blotting , Células da Medula Óssea/metabolismo , Proliferação de Células , Transformação Celular Neoplásica/metabolismo , Modelos Animais de Doenças , Feminino , Citometria de Fluxo , Perfilação da Expressão Gênica , Histonas/metabolismo , Humanos , Estimativa de Kaplan-Meier , Linfoma/metabolismo , Linfoma/patologia , Linfoma de Células B/genética , Linfoma de Células B/metabolismo , Linfoma de Células B/patologia , Lisina/metabolismo , Masculino , Metilação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Complexo Repressor Polycomb 2/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Baço/metabolismo , Baço/patologia
5.
N Engl J Med ; 366(3): 234-42, 2012 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-22187960

RESUMO

BACKGROUND: Germline truncating mutations in DICER1, an endoribonuclease in the RNase III family that is essential for processing microRNAs, have been observed in families with the pleuropulmonary blastoma-family tumor and dysplasia syndrome. Mutation carriers are at risk for nonepithelial ovarian tumors, notably sex cord-stromal tumors. METHODS: We sequenced the whole transcriptomes or exomes of 14 nonepithelial ovarian tumors and noted closely clustered mutations in the region of DICER1 encoding the RNase IIIb domain of DICER1 in four samples. We then sequenced this region of DICER1 in additional ovarian tumors and in certain other tumors and queried the effect of the mutations on the enzymatic activity of DICER1 using in vitro RNA cleavage assays. RESULTS: DICER1 mutations in the RNase IIIb domain were found in 30 of 102 nonepithelial ovarian tumors (29%), predominantly in Sertoli-Leydig cell tumors (26 of 43, or 60%), including 4 tumors with additional germline DICER1 mutations. These mutations were restricted to codons encoding metal-binding sites within the RNase IIIb catalytic centers, which are critical for microRNA interaction and cleavage, and were somatic in all 16 samples in which germline DNA was available for testing. We also detected mutations in 1 of 14 nonseminomatous testicular germ-cell tumors, in 2 of 5 embryonal rhabdomyosarcomas, and in 1 of 266 epithelial ovarian and endometrial carcinomas. The mutant DICER1 proteins had reduced RNase IIIb activity but retained RNase IIIa activity. CONCLUSIONS: Somatic missense mutations affecting the RNase IIIb domain of DICER1 are common in nonepithelial ovarian tumors. These mutations do not obliterate DICER1 function but alter it in specific cell types, a novel mechanism through which perturbation of microRNA processing may be oncogenic. (Funded by the Terry Fox Research Institute and others.).


Assuntos
RNA Helicases DEAD-box/genética , Mutação de Sentido Incorreto , Neoplasias Ovarianas/genética , Ribonuclease III/genética , Tumor de Células de Sertoli-Leydig/genética , Carcinossarcoma/genética , Feminino , Expressão Gênica , Perfilação da Expressão Gênica , Mutação em Linhagem Germinativa , Humanos , MicroRNAs/metabolismo , Neoplasias Embrionárias de Células Germinativas/genética , Rabdomiossarcoma/genética , Análise de Sequência de DNA
6.
Blood ; 117(8): 2451-9, 2011 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-21190999

RESUMO

Next-generation sequencing of follicular lymphoma and diffuse-large B-cell lymphoma has revealed frequent somatic, heterozygous Y641 mutations in the histone methyltransferase EZH2. Heterozygosity and the presence of equal quantities of both mutant and wild-type mRNA and expressed protein suggest a dominant mode of action. Surprisingly, B-cell lymphoma cell lines and lymphoma samples harboring heterozygous EZH2(Y641) mutations have increased levels of histone H3 Lys-27-specific trimethylation (H3K27me3). Expression of EZH2(Y641F/N) mutants in cells with EZH2(WT) resulted in an increase of H3K27me3 levels in vivo. Structural modeling of EZH2(Y641) mutants suggests a "Tyr/Phe switch" model whereby structurally neutral, nontyrosine residues at position 641 would decrease affinity for unmethylated and monomethylated H3K27 substrates and potentially favor trimethylation. We demonstrate, using in vitro enzyme assays of reconstituted PRC2 complexes, that Y641 mutations result in a decrease in monomethylation and an increase in trimethylation activity of the enzyme relative to the wild-type enzyme. This represents the first example of a disease-associated gain-of-function mutation in a histone methyltransferase, whereby somatic EZH2 Y641 mutations in lymphoma act dominantly to increase, rather than decrease, histone methylation. The dominant mode of action suggests that allele-specific EZH2 inhibitors should be a future therapeutic strategy for this disease.


Assuntos
Proteínas de Ligação a DNA/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Linfoma Difuso de Grandes Células B/genética , Mutação de Sentido Incorreto , Fatores de Transcrição/genética , Biópsia , Catálise , Linhagem Celular Tumoral , Proteína Potenciadora do Homólogo 2 de Zeste , Humanos , Linfoma/genética , Metilação , Modelos Moleculares , Complexo Repressor Polycomb 2 , Especificidade por Substrato
7.
PLoS Pathog ; 5(6): e1000463, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19503603

RESUMO

HIV-1 integrase (IN) is a virally encoded protein required for integration of viral cDNA into host chromosomes. INI1/hSNF5 is a component of the SWI/SNF complex that interacts with HIV-1 IN, is selectively incorporated into HIV-1 (but not other retroviral) virions, and modulates multiple steps, including particle production and infectivity. To gain further insight into the role of INI1 in HIV-1 replication, we screened for INI1-interacting proteins using the yeast two-hybrid system. We found that SAP18 (Sin3a associated protein 18 kD), a component of the Sin3a-HDAC1 complex, directly binds to INI1 in yeast, in vitro and in vivo. Interestingly, we found that IN also binds to SAP18 in vitro and in vivo. SAP18 and components of a Sin3A-HDAC1 complex were specifically incorporated into HIV-1 (but not SIV and HTLV-1) virions in an HIV-1 IN-dependent manner. Using a fluorescence-based assay, we found that HIV-1 (but not SIV) virion preparations harbour significant deacetylase activity, indicating the specific recruitment of catalytically active HDAC into the virions. To determine the requirement of virion-associated HDAC1 to HIV-1 replication, an inactive, transdominant negative mutant of HDAC1 (HDAC1(H141A)) was utilized. Incorporation of HDAC1(H141A) decreased the virion-associated histone deacetylase activity. Furthermore, incorporation of HDAC1(H141A) decreased the infectivity of HIV-1 (but not SIV) virions. The block in infectivity due to virion-associated HDAC1(H141A) occurred specifically at the early reverse transcription stage, while entry of the virions was unaffected. RNA-interference mediated knock-down of HDAC1 in producer cells resulted in decreased virion-associated HDAC1 activity and a reduction in infectivity of these virions. These studies indicate that HIV-1 IN and INI1/hSNF5 bind SAP18 and selectively recruit components of Sin3a-HDAC1 complex into HIV-1 virions. Furthermore, HIV-1 virion-associated HDAC1 is required for efficient early post-entry events, indicating a novel role for HDAC1 during HIV-1 replication.


Assuntos
Proteínas de Transporte/metabolismo , Integrase de HIV/metabolismo , HIV-1/fisiologia , Histona Desacetilases/metabolismo , Replicação Viral , Linhagem Celular , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Correpressoras , Proteínas de Ligação a DNA/metabolismo , Interpretação Estatística de Dados , HIV-1/metabolismo , Histona Desacetilase 1 , Histona Desacetilases/genética , Humanos , Imunoprecipitação , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA , Proteínas Repressoras/metabolismo , Proteína SMARCB1 , Vírus da Imunodeficiência Símia/metabolismo , Complexo Correpressor Histona Desacetilase e Sin3 , Fatores de Transcrição/metabolismo , Vírion/metabolismo
8.
Cancer Res ; 80(17): 3480-3491, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32641414

RESUMO

The somatic missense point mutation c.402C>G (p.C134W) in the FOXL2 transcription factor is pathognomonic for adult-type granulosa cell tumors (AGCT) and a diagnostic marker for this tumor type. However, the molecular consequences of this mutation and its contribution to the mechanisms of AGCT pathogenesis remain unclear. To explore these mechanisms, we engineered V5-FOXL2WT- and V5-FOXL2C134W-inducible isogenic cell lines and performed chromatin immunoprecipitation sequencing and transcriptome profiling. FOXL2C134W associated with the majority of the FOXL2 wild-type DNA elements as well as a large collection of unique elements genome wide. This model enabled confirmation of altered DNA-binding specificity for FOXL2C134W and identification of unique targets of FOXL2C134W including SLC35F2, whose expression increased sensitivity to YM155. Our results suggest FOXL2C134W drives AGCT by altering the binding affinity of FOXL2-containing complexes to engage an oncogenic transcriptional program. SIGNIFICANCE: A mechanistic understanding of FOXL2C134W-induced regulatory state alterations drives discovery of a rationally designed therapeutic strategy.


Assuntos
DNA/metabolismo , Proteína Forkhead Box L2/genética , Proteína Forkhead Box L2/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Tumor de Células da Granulosa/genética , Linhagem Celular Tumoral , Feminino , Tumor de Células da Granulosa/metabolismo , Humanos , Mutação de Sentido Incorreto , Mutação Puntual , Ligação Proteica
9.
PLoS Biol ; 3(2): e44, 2005 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15719058

RESUMO

The human immunodeficiency virus type I (HIV-1) transactivator protein Tat is an unusual transcriptional activator that is thought to act solely by promoting RNA polymerase II processivity. Here we study the mechanism of Tat action by analyzing transcription complex (TC) assembly in vivo using chromatin immunoprecipitation assays. We find, unexpectedly, that like typical activators Tat dramatically stimulates TC assembly. Surprisingly, however, the TC formed on the HIV-1 long terminal repeat is atypical and contains TATA-box-binding protein (TBP) but not TBP-associated factors (TAFs). Tat function involves direct interaction with the cellular cofactor positive transcription elongation factor b (P-TEFb). Artificial tethering of P-TEFb subunits to HIV-1 promoter DNA or nascent RNA indicates that P-TEFb is responsible for directing assembly of a TC containing TBP but not TAFs. On the basis of this finding, we identify P-TEFb-dependent cellular promoters that also recruit TBP in the absence of TAFs. Thus, in mammalian cells transcription of protein-coding genes involves alternative TCs that differ by the presence or absence of TAFs.


Assuntos
Produtos do Gene tat/metabolismo , HIV-1/genética , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Proteína de Ligação a TATA-Box/metabolismo , Transcrição Gênica , Animais , Globinas/genética , Células HeLa , Humanos , Regiões Promotoras Genéticas , Proteínas Recombinantes/metabolismo , Fatores Associados à Proteína de Ligação a TATA/genética , Proteína de Ligação a TATA-Box/genética , Transfecção , Produtos do Gene tat do Vírus da Imunodeficiência Humana
10.
Nat Commun ; 8(1): 7, 2017 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-28232751

RESUMO

CDC-like kinase phosphorylation of serine/arginine-rich proteins is central to RNA splicing reactions. Yet, the genomic network of CDC-like kinase-dependent RNA processing events remains poorly defined. Here, we explore the connectivity of genomic CDC-like kinase splicing functions by applying graduated, short-exposure, pharmacological CDC-like kinase inhibition using a novel small molecule (T3) with very high potency, selectivity, and cell-based stability. Using RNA-Seq, we define CDC-like kinase-responsive alternative splicing events, the large majority of which monotonically increase or decrease with increasing CDC-like kinase inhibition. We show that distinct RNA-binding motifs are associated with T3 response in skipped exons. Unexpectedly, we observe dose-dependent conjoined gene transcription, which is associated with motif enrichment in the last and second exons of upstream and downstream partners, respectively. siRNA knockdown of CLK2-associated genes significantly increases conjoined gene formation. Collectively, our results reveal an unexpected role for CDC-like kinase in conjoined gene formation, via regulation of 3'-end processing and associated splicing factors.The phosphorylation of serine/arginine-rich proteins by CDC-like kinase is a central regulatory mechanism for RNA splicing reactions. Here, the authors synthesize a novel small molecule CLK inhibitor and map CLK-responsive alternative splicing events and discover an effect on conjoined gene transcription.


Assuntos
Processamento Alternativo/efeitos dos fármacos , Imidazóis/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Pirimidinas/farmacologia , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Éxons , Perfilação da Expressão Gênica , Genoma Humano , Células HCT116 , Humanos , Imidazóis/síntese química , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/síntese química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Pirimidinas/síntese química , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/metabolismo , Relação Estrutura-Atividade , Transcrição Gênica
11.
Sci Rep ; 5: 17122, 2015 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-26597175

RESUMO

Cyclin-dependent kinase 12 (CDK12) promotes transcriptional elongation by phosphorylation of the RNA polymerase II C-terminal domain (CTD). Structure-function studies show that this activity is dependent on a C-terminal kinase extension, as well as the binding of cyclin K (CycK). To better define these interactions we determined the crystal structure of the human CDK12/CycK complex with and without the kinase extension in the presence of AMP-PNP. The structures revealed novel features for a CDK, including a large ß4-ß5 loop insertion that contributes to the N-lobe interaction with the cyclin. We also observed two different conformations of the C-terminal kinase extension that effectively open and close the ATP pocket. Most notably, bound AMP-PNP was only observed when trapped in the closed state. Truncation of this C-terminal structure also diminished AMP-PNP binding, as well as the catalytic activity of the CDK12/CycK complex. Further kinetic measurements showed that the full length CDK12/CycK complex was significantly more active than the two crystallised constructs suggesting a critical role for additional domains. Overall, these results demonstrate the intrinsic flexibility of the C-terminal extension in CDK12 and highlight its importance for both ATP binding and kinase activity.


Assuntos
Adenilil Imidodifosfato/química , Quinases Ciclina-Dependentes/química , Ciclinas/química , Animais , Domínio Catalítico , Cristalografia por Raios X , Humanos , Ligação de Hidrogênio , Modelos Moleculares , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Células Sf9 , Solubilidade , Spodoptera
12.
Mol Cell Biol ; 32(22): 4691-704, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22988298

RESUMO

CrkRS (Cdc2-related kinase, Arg/Ser), or cyclin-dependent kinase 12 (CKD12), is a serine/threonine kinase believed to coordinate transcription and RNA splicing. While CDK12/CrkRS complexes were known to phosphorylate the C-terminal domain (CTD) of RNA polymerase II (RNA Pol II), the cyclin regulating this activity was not known. Using immunoprecipitation and mass spectrometry, we identified a 65-kDa isoform of cyclin K (cyclin K1) in endogenous CDK12/CrkRS protein complexes. We show that cyclin K1 complexes isolated from mammalian cells contain CDK12/CrkRS but do not contain CDK9, a presumed partner of cyclin K. Analysis of extensive RNA-Seq data shows that the 65-kDa cyclin K1 isoform is the predominantly expressed form across numerous tissue types. We also demonstrate that CDK12/CrkRS is dependent on cyclin K1 for its kinase activity and that small interfering RNA (siRNA) knockdown of CDK12/CrkRS or cyclin K1 has similar effects on the expression of a luciferase reporter gene. Our data suggest that cyclin K1 is the primary cyclin partner for CDK12/CrkRS and that cyclin K1 is required to activate CDK12/CrkRS to phosphorylate the CTD of RNA Pol II. These properties are consistent with a role of CDK12/CrkRS in regulating gene expression through phosphorylation of RNA Pol II.


Assuntos
Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Regulação da Expressão Gênica , RNA Polimerase II/metabolismo , Sítios de Ligação , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/genética , Ciclinas/antagonistas & inibidores , Ciclinas/genética , Genes Reporter , Células HEK293 , Células HeLa , Humanos , Imunoprecipitação , Luciferases , Espectrometria de Massas , Fosforilação , Ligação Proteica , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , RNA Polimerase II/genética , RNA Interferente Pequeno/genética , Transdução de Sinais/genética
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