Objective To prepare rabbit polyclonal antibody against mouse IQ and ubiquitin-like domain-containing protein (IQUB) and detect its expression in the mouse testis. Methods Full-length coding sequence of IQUB was inserted into the pET-30a(+) vector to construct pET-30a-IQUB recombinant prokaryotic plasmid. Transformation of pET-30a-IQUB plasmid into E. coli BL21 was performed, and protein expression was induced with isopropyl-beta-D-thiogalactoside (IPTG). The protein was purified through histidine-tagged fusion protein purification column, then denatured by treatment of urea with gradient concentration. New Zealand rabbits were immunized with the denatured protein to produce IQUB polyclonal antibody. Antibody titer was detected by ELISA, and Western blot analysis and immunofluorescence assay were employed to validate the effectiveness and specificity of IQUB antibody. Results pET-30a-IQUB recombinant plasmid was constructed, and protein expression of IQUB was induced successfully with IPTG. The titer of IQUB polyclonal antibody reached 1:1 000 000. The antibody specifically recognized the endogenous IQUB protein of testis in the wild-type adult mouse. IQUB was expressed in spermatogenic cells of different stages. It was localized in the acrosome and flagellum of mature sperms. Conclusion The highly specific rabbit anti-mouse IQUB polyclonal antibody is successfully prepared, which can be used for Western blot and immunofluorescence histochemistry.
Male ; Rabbits ; Animals ; Mice ; Ubiquitins ; Escherichia coli/genetics* ; Isopropyl Thiogalactoside ; Antibodies ; Enzyme-Linked Immunosorbent Assay

Brain development requires a delicate balance between self-renewal and differentiation in neural stem cells (NSC), which rely on the precise regulation of gene expression. Ten-eleven translocation 2 (TET2) modulates gene expression by the hydroxymethylation of 5-methylcytosine in DNA as an important epigenetic factor and participates in the neuronal differentiation. Yet, the regulation of TET2 in the process of neuronal differentiation remains unknown. Here, the protein level of TET2 was reduced by the ubiquitin-proteasome pathway during NSC differentiation, in contrast to mRNA level. We identified that TET2 physically interacts with the core subunits of the glucose-induced degradation-deficient (GID) ubiquitin ligase complex, an evolutionarily conserved ubiquitin ligase complex and is ubiquitinated by itself. The protein levels of GID complex subunits increased reciprocally with TET2 level upon NSC differentiation. The silencing of the core subunits of the GID complex, including WDR26 and ARMC8, attenuated the ubiquitination and degradation of TET2, increased the global 5-hydroxymethylcytosine levels, and promoted the differentiation of the NSC. TET2 level increased in the brain of the Wdr26^+/- mice. Our results illustrated that the GID complex negatively regulates TET2 protein stability, further modulates NSC differentiation, and represents a novel regulatory mechanism involved in brain development.
Animals ; Mice ; DNA-Binding Proteins/genetics* ; Cell Differentiation ; Neural Stem Cells ; Translocation, Genetic ; Ubiquitins/genetics* ; Ligases/genetics*

OBJECTIVETo investigate whether progesterone receptor B (PRB) can be sumoylated by SUMO-2/3 and the effect of sumoylation on PRB transcriptional activity.

METHODSSUMO-2/3 cDNA was amplified from MCF-7 cDNA and cloned into the eukaryotic expression vector pcDNA3-FLAG. The plasmid pXJ40-myc-PRB was cotransfected with pcDNA3FLAG-SUMO2, pcDNA3FLAG-SUMO3 or the mock control into 293T cells, and PRB sumoylation was detected by immunoprecipitation and Western blotting. The effect of PRB sumoylation on its transcriptional activity was determined using reporter luciferase assay.

RESULTSpcDNA3FLAG-SUMO2 and pcDNA3FLAG-SUMO3 vectors were successfully constructed. SUMO-2/3 could bind covalently to PRB and increase its transcriptional dependent on the presence of progesterone.

CONCLUSIONPRB can be sumoylated by SUMO-2/3 and its function is regulated by this modification.

Animals ; Cell Line ; Humans ; Plasmids ; genetics ; Receptors, Progesterone ; genetics ; metabolism ; Small Ubiquitin-Related Modifier Proteins ; genetics ; metabolism ; Transcription, Genetic ; Transfection ; Ubiquitination ; Ubiquitins ; genetics ; metabolism

To investigate whether HIV-1 infection affects expression of interferon-stimulated gene 15 (ISG15) and determine the antiviral effect of ISG15 in vitro, ISG15 expression at transcription and protein level and supernatant p24 of HIV-1 was detected in various HIV-1 infected or transfected cell lines, respec tively. HIV-1 molecular clone pNL4-3 was used to transfect 293T, TZM-bl and HeLa cells while HIV-1 pseudo-typed virus was used to infect Jurkat, MT-1 and THP-1 cells. After twenty-four hours post infection or transfection, cells were harvested for extraction of total RNAs and subsequently used in real time PCR for quantification of ISG15 transcriptional expression. After forty-eight hours post infection or transfection, cells were harvested for extraction of total proteins to detect ISG15 protein expression. A significant up-regulation of ISG15 at transcription level was observed in HIV-1 infected or transfected cell lines, particulaly in THP-1 and TZM-bl cells. Up-regulation of ISG15 protein was observed only in TZM-bl cell. Cotransfection of ISG15 and HIV-1 indicated that ISG15 inhibited production of HIV-1 progeny virus in a dose and time depend manner in 293T cell but not TZM-bl cell. These results revealed upregulating ISG15 expression in transcriptional level and potential antagonistic mechanism against ISG15 by HIV-1 infection, simultanelusly.
Base Sequence ; Cell Line ; Cytokines ; genetics ; metabolism ; HIV Infections ; genetics ; metabolism ; virology ; HIV-1 ; physiology ; Humans ; Interferons ; metabolism ; Molecular Sequence Data ; Ubiquitins ; genetics ; metabolism ; Up-Regulation

OBJECTIVETo investigate the association between small ubiquitin-related modifier-1 (SUMO-1) gene rs6709162, rs7599810, rs7580433 polymorphism and non-syndromic oral clefting (NSOC).

METHODSOur study consisted of 208 Ningxia NSOC patients, their parents (189 fathers and 176 mothers), 172 nuclear families (patients and their parents), and 284 normal controls. DNA was extracted and polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) was used to identify rs6709162, rs7599810, rs7580433 genotypes of the samples. The data was analyzed by case-control analysis, family based associated test (FBAT), and transmission disequilibrium test (TDT).

RESULTSCase-control study found that TT genotype's frequency was significantly different in cleft lip and cleft palate group compared with the control group at rs7599810 of SUMO-1 (P=0.01, P=0.01). TDT test showed that rs7599810's T allele had over-transmitted (P=0.00) in cleft lip and palate group. FBAT analysis revealed that distribution of rs7599810's TT genotype and T allele was significantly different (P=0.00, P=0.00). TDT test showed that rs6709162's C allele in cleft palate and cleft lip and palate patients had over-transmitted (P=0.00, P=0.01). rs7580433's G allele in cleft lip group had over-transmitted (P=0.05).

CONCLUSIONSUMO-1 gene polymorphism is associated with NSOC.

Case-Control Studies ; Cerebellar Ataxia ; Cleft Lip ; Cleft Palate ; Female ; Genotype ; Humans ; Intellectual Disability ; Male ; Polymerase Chain Reaction ; Polymorphism, Genetic ; SUMO-1 Protein ; genetics ; Ubiquitins

OBJECTIVETo study the effect of diubiquitin (FAT10) down-regulation by small interfering RNA-mediated RNA interference (RNAi) on the biological features of tongue carcinoma cell line Tca8113.

METHODSTca8113 cells were transfected with synthetic small interfering RNA (siRNA) targeting FAT10. Expression of FAT10 mRNA and protein were respectively measured by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting, transfection efficiencies were monitored. The distribution of cell cycle phases was determined using flow cytometry. The proliferative and invasive ability of Tca8113 cells in vitro was evaluated by the colony-forming unit assay and Transwell migration assay respectively.

RESULTSBoth FAT10 mRNA and protein expression were significantly decreased in the experimental group (pU-FAT10-siRNA: mRAN 0.36 ± 0.03, Protein 0.39 ± 0.04) compared with controls (

CONTROLmRNA 0.95 ± 0.05, Protein 0.69 ± 0.05; pU-siRNA: mRNA 0.92 ± 0.07, Protein 0.64 ± 0.05) (P < 0.05). The cell cycle was arrested in the G(1) phase [pU-FAT10-siRNA: (72.45 ± 5.81)%,

CONTROL(45.95 ± 3.80)%, pU-siRNA: (45.95 ± 3.80)%]. The proliferation and invasiveness of treated Tca8113 cells were inhibited in vitro (pU-FAT10-siRNA: 41.83 ± 8.19, CONTROL: 317.21 ± 69.48, pU-siRNA: 339.36 ± 73.84).

CONCLUSIONSDelivery of siRNA targeting FAT10 seems efficient in down-regulating FAT10 expression and diminishing the growth, proliferation and invasiveness of Tca8113 cells, suggesting that siRNA-based strategy targeting FAT10 may lay a foundation for the clinical management of tongue carcinoma.

Carcinoma, Squamous Cell ; genetics ; metabolism ; pathology ; Cell Cycle Checkpoints ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Down-Regulation ; Humans ; RNA Interference ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; Tongue Neoplasms ; genetics ; metabolism ; pathology ; Transfection ; Ubiquitins ; genetics ; metabolism

OBJECTIVETo isolate and identify the differentially expressed genes in spermatogenesis for the understanding molecular mechanism of spermatogenesis.

METHODSScreening of the cDNA library, Northern blot, expression and purification in E. coli with GST expression system, immunocytochemical staining of testis sections were used.

RESULTS(1) A cDNA fragment designated as RSD-7 was isolated from rat testis cDNA library. It was 1,238 bp in length, coding a protein of 232 amino acids with the GenBank accession number AF315467. The encoding protein of RSD-7 cDNA had a Ubiquitin-like domain. (2) Northern blot indicated that RSD-7 was uniquely expressed in rat testis, and in the testis RSD-7 emerged on the 30th postnatal day and expressed until 120th postnatal day. (3) Expression and purification of RSD-7 protein in E. coli with GST expression system and were used to obtain anti-RSD-7 antibody. (4) Immunolocalization of RSD-7 in rat testis revealed that it is expressed only in Sertoli cells.

CONCLUSIONSTranscription pattern of RSD-7 and localization of RSD-7 protein in testis have been made, which established the base for the functional study of RSD-7.

Amino Acid Sequence ; Animals ; Base Sequence ; Cloning, Molecular ; DNA, Complementary ; biosynthesis ; genetics ; Escherichia coli ; genetics ; Escherichia coli Proteins ; biosynthesis ; genetics ; Male ; Molecular Sequence Data ; Rabbits ; Rats ; Rats, Wistar ; Repressor Proteins ; biosynthesis ; genetics ; Sertoli Cells ; metabolism ; Spermatogenesis ; Testis ; metabolism ; Ubiquitins ; biosynthesis ; genetics

SUMOylation is recently found to function as a targeting signal for the degradation of substrates through the ubiquitin-proteasome system. RNF4 is the most studied human SUMO-targeted ubiquitin E3 ligase. However, the relationship between SUMO proteases, SENPs, and RNF4 remains obscure. There are limited examples of the SENP regulation of SUMO2/3-targeted proteolysis mediated by RNF4. The present study investigated the role of SENP3 in the global protein turnover related to SUMO2/3-targeted ubiquitination and focused in particular on the SENP3 regulation of the stability of Sp1. Our data demonstrated that SENP3 impaired the global ubiquitination profile and promoted the accumulation of many proteins. Sp1, a cancer-associated transcription factor, was among these proteins. SENP3 increased the level of Sp1 protein via antagonizing the SUMO2/3-targeted ubiquitination and the consequent proteasome-dependent degradation that was mediated by RNF4. De-conjugation of SUMO2/3 by SENP3 attenuated the interaction of Sp1 with RNF4. In gastric cancer cell lines and specimens derived from patients and nude mice, the level of Sp1 was generally increased in parallel to the level of SENP3. These results provided a new explanation for the enrichment of the Sp1 protein in various cancers, and revealed a regulation of SUMO2/3 conjugated proteins whose levels may be tightly controlled by SENP3 and RNF4.
Animals ; Cysteine Endopeptidases ; genetics ; metabolism ; Gene Expression Regulation, Neoplastic ; Humans ; Immunoenzyme Techniques ; Immunoprecipitation ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Prognosis ; Protein Processing, Post-Translational ; Proteolysis ; RNA, Messenger ; genetics ; Real-Time Polymerase Chain Reaction ; Reverse Transcriptase Polymerase Chain Reaction ; Small Ubiquitin-Related Modifier Proteins ; antagonists & inhibitors ; genetics ; metabolism ; Sp1 Transcription Factor ; genetics ; metabolism ; Stomach Neoplasms ; genetics ; metabolism ; pathology ; Sumoylation ; Tumor Cells, Cultured ; Ubiquitination ; Ubiquitins ; antagonists & inhibitors ; genetics ; metabolism ; Xenograft Model Antitumor Assays

OBJECTIVETo define the differences in gene expression patterns between glycidyl methacrylate (GMA)-transformed human lung fibroblast cells (2BS cells) and controls.

METHODSThe mRNA differential display polymerase chain reaction (DD-PCR) technique was used. cDNAs were synthesized by reverse transcription and amplified by PCR using 30 primer combinations. After being screened by dot blot analysis, differentially expressed cDNAs were cloned, sequenced and confirmed by Northern blot analysis.

RESULTSEighteen differentially expressed cDNAs were cloned and sequenced, of which 17 were highly homologous to known genes (homology = 89%-100%) and one was an unknown gene. Northern blot analysis confirmed that eight genes encoding human zinc finger protein 217 (ZNF217), mixed-lineage kinase 3 (MLK-3), ribosomal protein (RP) L15, RPL41, RPS 16, TBX3, stanniocalcin 2 (STC2) and mouse ubiquitin conjugating enzyme (UBC), respectively, were up-regulated, and three genes including human transforming growth factor beta inducible gene (Betaig-h3), alpha-1,2-mannosidase 1A2 (MAN 1A2) gene and an unknown gene were down-regulated in the GMA-transformed cells.

CONCLUSIONAnalysis of the potential function of these genes suggest that they may be possibly linked to a variety of cellular processes such as transcription, signal transduction, protein synthesis and growth, and that their differential expression could contribute to the GMA-induced neoplastic transformation.

Air Pollutants, Occupational ; toxicity ; Carcinoma, Squamous Cell ; genetics ; pathology ; Cell Line, Transformed ; Epoxy Compounds ; toxicity ; Fibroblasts ; cytology ; drug effects ; Gene Expression Profiling ; Glycoproteins ; metabolism ; Humans ; Lung ; cytology ; Male ; Mannosidases ; drug effects ; metabolism ; Methacrylates ; toxicity ; Mitogen-Activated Protein Kinase 3 ; drug effects ; metabolism ; Oligonucleotide Array Sequence Analysis ; Ribosomal Proteins ; metabolism ; Signal Transduction ; genetics ; Transforming Growth Factor beta ; drug effects ; metabolism ; Ubiquitins ; metabolism ; Zinc Fingers ; drug effects ; physiology