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  • Oncogenicity of the developmental transcription factor Sox9. 22246670

    SOX9 [sex-determining region Y (SRY)-box 9 protein], a high mobility group box transcription factor, plays critical roles during embryogenesis and its activity is required for development, differentiation, and lineage commitment in various tissues including the intestinal epithelium. Here, we present functional and clinical data of a broadly important role for SOX9 in tumorigenesis. SOX9 was overexpressed in a wide range of human cancers, where its expression correlated with malignant character and progression. Gain of SOX9 copy number is detected in some primary colorectal cancers. SOX9 exhibited several pro-oncogenic properties, including the ability to promote proliferation, inhibit senescence, and collaborate with other oncogenes in neoplastic transformation. In primary mouse embryo fibroblasts and colorectal cancer cells, SOX9 expression facilitated tumor growth and progression whereas its inactivation reduced tumorigenicity. Mechanistically, we have found that Sox9 directly binds and activates the promoter of the polycomb Bmi1, whose upregulation represses the tumor suppressor Ink4a/Arf locus. In agreement with this, human colorectal cancers showed a positive correlation between expression levels of SOX9 and BMI1 and a negative correlation between SOX9 and ARF in clinical samples. Taken together, our findings provide direct mechanistic evidence of the involvement of SOX9 in neoplastic pathobiology, particularly, in colorectal cancer.
    Document Type:
    Reference
    Product Catalog Number:
    AB5535
    Product Catalog Name:
    Anti-Sox9 Antibody

  • Conditional deletion of the retinoblastoma (Rb) gene in ovarian granulosa cells leads to premature ovarian failure. 18599617

    The retinoblastoma protein (RB) regulates cell proliferation and survival by binding to the E2F family of transcription factors. Recent studies suggest that RB also regulates differentiation in a variety of cell types, including myocytes, neurons, adipocytes, and chondrocytes. Rb mutations have been found in ovarian cancer; however, the role of RB in normal and abnormal ovarian function remains unclear. To test the hypothesis that loss of Rb induces ovarian tumorigenesis, we generated an ovarian granulosa cell conditional knockout of Rb (Rb cKO) using the Cre/lox recombination system. Rb cKO females showed 100% survival and no ovarian tumor formation through 9 months of age, but they developed progressive infertility. Prepubertal Rb cKO females showed increased ovulation rates compared with controls, correlating with increased follicle recruitment, higher Fshr and Kitl mRNA levels, and lower anti-Müllerian hormone levels. In contrast, the ovulation rate of 6-wk-old females was similar to that of controls. Morphometric analysis of Rb cKO ovaries from 6-wk-old and older females showed increased follicular atresia and apoptosis. Rb cKO ovaries and preantral follicles had abnormal levels of known direct and indirect target genes of RB, including Rbl2/p130, E2f1, Ccne2, Myc, Fos, and Tgfb2. In addition, preantral follicles showed increased expression of the granulosa cell differentiation marker Inha, decreased levels of Foxl2 and Cyp19a1 aromatase, and abnormal expression of the nuclear receptors Nr5a1, Nr5a2, and Nr0b1. Taken together, our results suggest that RB is required for the temporal-specific pattern of expression of key genes involved in follicular development.
    Document Type:
    Reference
    Product Catalog Number:
    S7111
    Product Catalog Name:
    ApopTag® Plus In Situ Apoptosis Fluorescein Detection Kit

  • Rafs constitute a nodal point in the regulation of embryonic endothelial progenitor cell growth and differentiation. 18205709

    Mouse embryonic endothelial progenitor cells (eEPCs) acquire a mature phenotype after treatment with cyclic adenosine monophosphate (cAMP), suggesting an involvement of Raf serine/threonine kinases in the differentiation process. To test this idea, we investigated the role of B-Raf and C-Raf in proliferation and differentiation of eEPCs by expressing fusion proteins consisting of the kinase domains from Raf molecules and the hormone binding site of the estrogen receptor (ER), or its variant, the tamoxifen receptor. Our findings show that both B- and C-Raf kinase domains, when lacking adjacent regulatory parts, are equally effective in inducing eEPC differentiation. In contrast, the C-Raf kinase domain is a more potent stimulator of eEPC proliferation than B-Raf. In a complimentary approach, we used siRNA silencing to knockdown endogenously expressed B-Raf and C-Raf in eEPCs. In this experimental setting, we found that eEPCs lacking B-Raf failed to differentiate, whereas loss-of C-Raf function primarily slowed cell growth without impairing cAMP-induced differentiation. These findings were further corroborated in B-Raf null eEPCs, isolated from the corresponding knockout embryos, which failed to differentiate in vitro. Thus, gain- and loss-of-function experiments point to distinct roles of B-Raf and C-Raf in regulating growth and differentiation of endothelial progenitor cells, which may harbour therapeutic implications.
    Document Type:
    Reference
    Product Catalog Number:
    07-453
    Product Catalog Name:
    Anti-B-Raf Antibody

  • Identification of a dominant negative functional domain on DAPK-1 that degrades DAPK-1 protein and stimulates TNFR-1-mediated apoptosis. 17324927

    DAPK-1 is a stress-activated tumor suppressor protein that plays a role in both proapoptotic or antiapoptotic signal transduction pathways. To define mechanisms of DAPK-1 protein regulation, we have determined that DAPK-1 protein has a long half-life, and therefore its activity is primarily regulated at the protein level. Changes in DAPK-1 protein levels occur by a cathepsin B-dependent pathway, prompting us to evaluate whether cathepsin B plays positive or negative role in DAPK-1 function. The transfection of p55-TNFR-1 induced complex formation between DAPK-1 and cathepsin B. Depletion of cathepsin B protein using small interfering RNA stimulated TNFR-1 dependent apoptosis. The minimal binding region on DAPK-1 for cathepsin B was mapped to amino acids 836-947. The transfection of the DAPK-1-(836-947) miniprotein acted in a dominant negative manner inducing endogenous DAPK-1 protein degradation in a TNFR-1-dependent manner. These data suggest that DAPK-1 forms a multiprotein survival complex with cathepsin B countering the rate of TNFR-1-dependent apoptosis and highlights the importance of developing DAPK-1 inhibitors as agents to sensitize cells to stress-induced apoptosis.
    Document Type:
    Reference
    Product Catalog Number:
    S7111
    Product Catalog Name:
    ApopTag® Plus In Situ Apoptosis Fluorescein Detection Kit

  • Irgm1 (LRG-47), a regulator of cell-autonomous immunity, does not localize to mycobacterial or listerial phagosomes in IFN-γ-induced mouse cells. 23842753

    The IFN-inducible protein Irgm1 (LRG-47) belongs to the family of immunity-related GTPases that function in cell-autonomous resistance against intracellular pathogens in mice. Irgm1 deficiency is associated with a severe immunodeficiency syndrome. The protein has been variously interpreted as a direct effector molecule on bacterial phagosomes or on other organelles or as an inducer of autophagy. In this study, we re-examined one of these claims, namely that Irgm1 targets mycobacterial and listerial phagosomes. We found no colocalization of endogenous Irgm1, using two immunofluorescent staining techniques, either in fibroblasts or in macrophages. We demonstrated the predicted existence of two protein isoforms of Irgm1 derived from differential splicing and described immunological reagents for their detection. Both Irgm1 isoforms localize to the Golgi apparatus and weakly to mitochondria; however, only the long Irgm1 isoforms can be detected on endolysosomal membranes. Together with the previous observation that the general immunodeficiency phenotype of Irgm1(-/-) mice is reversed in Irgm1/Irgm3 double-deficient mice, our results argue against a direct effector function of Irgm1 at the bacterial phagosome. We discuss these findings in the context of evidence that Irgm1 functions as a negative regulator of other members of the immunity-related GTPase protein family.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Nuclear translation visualized by ribosome-bound nascent chain puromycylation. 22472439

    Whether protein translation occurs in the nucleus is contentious. To address this question, we developed the ribopuromycylation method (RPM), which visualizes translation in cells via standard immunofluorescence microscopy. The RPM is based on ribosome-catalyzed puromycylation of nascent chains immobilized on ribosomes by antibiotic chain elongation inhibitors followed by detection of puromycylated ribosome-bound nascent chains with a puromycin (PMY)-specific monoclonal antibody in fixed and permeabilized cells. The RPM correlates localized translation with myriad processes in cells and can be applied to any cell whose translation is sensitive to PMY. In this paper, we use the RPM to provide evidence for translation in the nucleoplasm and nucleolus, which is regulated by infectious and chemical stress.
    Document Type:
    Reference
    Product Catalog Number:
    MABE343
    Product Catalog Name:
    Anti-Puromycin Antibody, clone 12D10

  • Ultrasensitive measurement of huntingtin protein in cerebrospinal fluid demonstrates increase with Huntington disease stage and decrease following brain huntingtin suppre ... 26174131

    Quantitation of huntingtin protein in the brain is needed, both as a marker of Huntington disease (HD) progression and for use in clinical gene silencing trials. Measurement of huntingtin in cerebrospinal fluid could be a biomarker of brain huntingtin, but traditional protein quantitation methods have failed to detect huntingtin in cerebrospinal fluid. Using micro-bead based immunoprecipitation and flow cytometry (IP-FCM), we have developed a highly sensitive mutant huntingtin detection assay. The sensitivity of huntingtin IP-FCM enables accurate detection of mutant huntingtin protein in the cerebrospinal fluid of HD patients and model mice, demonstrating that mutant huntingtin levels in cerebrospinal fluid reflect brain levels, increasing with disease stage and decreasing following brain huntingtin suppression. This technique has potential applications as a research tool and as a clinical biomarker.
    Document Type:
    Reference
    Product Catalog Number:
    MAB2166
    Product Catalog Name:
    Anti-Huntingtin Protein Antibody, a.a. 181-810, clone 1HU-4C8

  • Identification of potential bladder cancer markers in urine by abundant-protein depletion coupled with quantitative proteomics. 23631828

    In this study, we evaluated the reproducibility of abundant urine protein depletion by hexapeptide-based library beads and an antibody-based affinity column using the iTRAQ technique. The antibody-based affinity-depletion approach, which proved superior, was then applied in conjunction with iTRAQ to discover proteins that were differentially expressed between pooled urine samples from hernia and bladder cancer patients. Several proteins, including seven apolipoproteins, TIM, SAA4, and proEGF were further verified in 111 to 203 individual urine samples from patients with hernia, bladder cancer, or kidney cancer. Six apolipoproteins (APOA1, APOA2, APOB, APOC2, APOC3, and APOE) were able to differentiate bladder cancer from hernia. SAA4 was significantly increased in bladder cancer subgroups, whereas ProEGF was significantly decreased in bladder cancer subgroups. Additionally, the combination of SAA4 and ProEGF exhibited higher diagnostic capacity (AUC=0.80 and p<0.001) in discriminating bladder cancer from hernia than either marker alone. Using MetaCore software to interpret global changes of the urine proteome caused by bladder cancer, we found that the most notable alterations were in immune-response/alternative complement and blood-coagulation pathways. This study confirmed the clinical significance of the urine proteome in the development of non-invasive biomarkers for the detection of bladder cancer.In this study, we evaluated the reproducibility of abundant urine protein depletion by hexapeptide-based library beads and an antibody-based affinity column using the iTRAQ technique. The antibody-based affinity-depletion approach, which proved superior, was then applied in conjunction with iTRAQ to discover proteins that were differentially expressed between pooled urine samples from hernia and bladder cancer patients. Several proteins, including seven apolipoproteins, TIM, SAA4, and proEGF were further verified in 111 to 203 individual urine samples from patients with hernia, bladder cancer, or kidney cancer. SAA4 was significantly increased in bladder cancer subgroups, whereas ProEGF was significantly decreased in bladder cancer subgroups. Additionally, the combination of SAA4 and ProEGF exhibited higher diagnostic capacity in discriminating bladder cancer from hernia than either marker alone. A marker panel composed by two novel biomarker candidates, SAA4 and proEGF, was first discovered and verified successfully using Western blotting. To the best of our knowledge, the associations of urinary SAA4 and proEGF with bladder tumor and kidney cancer have not been mentioned before. In the present study, we discovered and verified SAA4 and proEGF as potential bladder cancer biomarker for the first time.
    Document Type:
    Reference
    Product Catalog Number:
    APOMAG-62K
    Product Catalog Name:
    MILLIPLEX MAP Human Apolipoprotein Magnetic Bead Panel - Cardiovascular Disease Multiplex Assay

  • Monoclonal antibody detection of CD46 clustering beneath Neisseria gonorrhoeae microcolonies 16552073

    CD46 (membrane cofactor protein), a complement-regulatory protein that participates in innate and acquired immunity, also serves as a receptor for viral and bacterial pathogens. CD46 isoforms terminate in one of two cytoplasmic tails, Cyt1 or Cyt2, which differ in signaling and trafficking properties. Dissecting the functions of the two cytoplasmic tails in these cellular processes has been hampered by the absence of specific reagents. Here we report the construction of Cyt1- and Cyt2-specific monoclonal antibodies (MAbs). These MAbs recognize unique epitopes within the tails and can be used for immunofluorescence microscopy, immunoblotting, and immunoprecipitation. Studies of Neisseria gonorrhoeae-infected cells with the CD46 tail MAbs demonstrate the differential recruitment of Cyt1 and Cyt2 to the cortical plaque.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • A protein profile study to discriminate CIN lesions from normal cervical epithelium. 21573931

    Cervical intraepithelial neoplasia (CIN), a frequently encountered disease caused by Human Papilloma Virus (HPV) is often diagnosed in formaldehyde-fixed paraffin embedded (FFPE) punch biopsies. Since it is known that this procedure strongly affects the water-soluble proteins contained in the cervical tissue we decided to investigate whether a water-soluble protein-saving biopsy processing method can be used to support the diagnosis of normal and CIN.Cervical punch biopsies from 55 women were incubated for 24 h at 4°C in RPMI1640 medium for protein analysis prior to usual FFPE processing and p16 and Ki67-supported histologic consensus diagnosis was assessed. The biopsy supernatants were subjected to surface-enhanced laser desorption-ionization time of flight mass spectrometry (SELDI-TOF MS) for identifying differentially expressed proteins. Binary logistic regression and classification and regression trees (CART) were used to develop a classification model.The age of the patients ranged from 26 to 40 years (median 29.7). The consensus diagnoses were normal cervical tissue (n = 10) and CIN2-3 (n = 45). The mean protein concentration was 1.00 and 1.09 mg/ml in the normal and CIN2-3 group, respectively. The peak detection and clustering process resulted in 40 protein peaks. Many of these peaks differed between the two groups, but only three had independent discriminating power. The overall classification results were 88%.Water-soluble proteins sampled from punch biopsies are promising to assist the diagnosis of normal and CIN2-3.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple