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  • Detection of citrulline residues in deiminated proteins on polyvinylidene difluoride membrane. 1524220

    We have developed a new detection method of deiminated proteins on polyvinylidene difluoride membranes. Citrulline residues in enzymatically deiminated histones were modified by incubating with diacetyl monoxime and antipyrine in a strong acid mixture. The products were injected to rabbits, and the antibodies obtained were affinity-purified using a modified citrulline column. Sample proteins blotted to the membrane were modified in a similar manner and incubated successively with the purified antibody and an alkaline phosphatase-conjugated second antibody. Detection was performed using a chemiluminescent substrate. The method enabled detection of 3-10 fmol of citrulline residues dot blotted as deiminated model proteins. It visualized numerous rat pituitary soluble proteins that had been enzymatically deiminated and Western blotted to the membrane. The data suggest usefulness of the method for detecting deiminated proteins regardless of the backbone protein molecules. Search for deiminated proteins on the Western blots of various rat tissue homogenates detected a single band on that of spinal cord, another band on that of uterus, and multiple bands on those of skin and hair root. The bands in the former two tissue homogenates comigrated with glial fibrillary acidic protein and vimentin, respectively.
    Document Type:
    Reference
    Product Catalog Number:
    17-347

  • Diabetes-induced alterations of adenosine receptors expression level in rat liver. 17490639

    Diabetes mellitus is associated with metabolic, functional, and structural changes in the liver. Adenosine has been demonstrated to play an important regulatory role in the liver, and its action has been associated with all four adenosine receptors (ARs) subtypes. The goal of this study was to evaluate the impact of streptozotocin-induced diabetes on expression level of ARs in rat liver. Performed analyses (real-time PCR, Western blots) revealed detectable levels of mRNA and protein of A(1)-AR, A(2A)-AR, A(2B)-AR, and A(3)-AR in the rat liver. Development of diabetes resulted in a significant increase of A(2A)-AR and A(3)-AR mRNA levels. This was associated with elevated ARs protein content. The level of A(2B)-AR mRNA in diabetic liver decreased approximately 40% and was accompanied by 60% drop in A(2B)-AR protein in liver membranes. Diabetes did not affect the expression level of A(1)-AR in the liver. Administration of insulin for four days to diabetic rats resulted in returning of the ARs expression to the levels observed in liver of normal rat. The changes in ARs genes expression and receptors protein content could be related to some pathological changes taking place in diabetic liver. This might suggest involvement of ARs in pathogenesis of liver disease.
    Document Type:
    Reference
    Product Catalog Number:
    AB1589P
    Product Catalog Name:
    Anti-Adenosine A2b Receptor Antibody

  • Association of dystroglycan and laminin-2 coexpression with myelinogenesis in peripheral nerves. 16228655

    To provide clues to the biological functions of dystroglycan-laminin-2 complex in peripheral nerves, we investigated the expressions of beta-dystroglycan and laminin-alpha(2) chain in rat sciatic nerve during axonal degeneration and regeneration and during development, as well as in rat dorsal root ganglia. In normal conditions, immunoreactivity of the cytoplasmic domain of beta-dystroglycan was associated with the Schwann cell abaxonal membrane. The immunoreactivities of both beta-dystroglycan and the laminin-alpha(2) chain decreased in Schwann cells losing axons during axonal degeneration and progressively increased in remyelinating Schwann cells during axonal regeneration. Interestingly, during axonal degeneration, the abaxonal membrane losing contact with the basal lamina lost the association with beta-dystroglycan immunoreactivity. During development, expression of both beta-dystroglycan and laminin-alpha(2) chain strikingly increased during postnatal 7 days, which is a critical period when basal lamina assembly and myelin formation rapidly progress. These results suggest that coexpression of dystroglycan and laminin-2 is associated with myelinogenesis in peripheral nerves. These two proteins may function as an anchorage between the abaxonal membrane and the basal lamina, enabling myelin forma-tion to progress. Beta-dystroglycan and laminin-2 were also coexpressed in satellite cells in dorsal root ganglia, suggesting that interaction of these two proteins plays some role in physiological functions of these cells.
    Document Type:
    Reference
    Product Catalog Number:
    AP182R
    Product Catalog Name:
    Donkey Anti-Rabbit IgG Antibody, Rhodamine conjugate, Species Adsorbed

  • Thyroid hormone, retinoic acid, and testosterone suppress proliferation and induce markers of differentiation in cultured rat sertoli cells. 12933640

    This study uses a high purity cell culture system to extend previous observations of factors controlling the end of the Sertoli cell proliferative phase. Thyroid hormone, retinoic acid, and testosterone were assessed for their ability to halt the proliferative phase and regulate the expression of markers associated with maturation of the Sertoli cell. We show that these hormones share similar suppressive effects on the rate of Sertoli cell division without any apparent additive effects. We demonstrate that these hormones induce the progressive accumulation of cell cycle inhibitors p27Kip1 and p21Cip1 in Sertoli cells, a likely regulatory mechanism controlling the suppression of proliferation. We used real-time RT-PCR to examine the effects of these factors on the expression of mRNA encoding the Id proteins, demonstrating an increase in Id2 and Id3 expression in Sertoli cells treated with thyroid hormone, retinoic acid, or testosterone. Finally, we examined the expression of a number of genes that have been implicated in the Sertoli cell differentiation process. Our results suggest that these hormones can induce aspects of Sertoli cell differentiation in vitro, providing a valuable in vitro model for studying Sertoli cell function.
    Document Type:
    Reference
    Product Catalog Number:
    MAB3408
    Product Catalog Name:
    Anti-Tubulin Antibody, beta, clone KMX-1

  • Sensitivity of cancer cells to Plk1 inhibitor GSK461364A is associated with loss of p53 function and chromosome instability. 20571075

    Polo-like kinases are a family of serine threonine kinases that are critical regulators of cell cycle progression and DNA damage response. Predictive biomarkers for the Plk1-selective inhibitor GSK461364A were identified by comparing the genomics and genetics of a panel of human cancer cell lines with their response to a drug washout followed by an outgrowth assay. In this assay, cell lines that have lost p53 expression or carry mutations in the TP53 gene tended to be more sensitive to GSK461364A. These more sensitive cell lines also had increased levels of chromosome instability, a characteristic associated with loss of p53 function. Further mechanistic studies showed that p53 wild-type (WT) and not mutant cells can activate a postmitotic tetraploidy checkpoint and arrest at pseudo-G(1) state after GSK461364A treatment. RNA silencing of WT p53 increased the antiproliferative activity of GSK461364A. Furthermore, silencing of p53 or p21/CDKN1A weakened the tetraploidy checkpoint in cells that survived mitotic arrest and mitotic slippage. As many cancer therapies tend to be more effective in p53 WT patients, the higher sensitivity of p53-deficient tumors toward GSK461364A could potentially offer an opportunity to treat tumors that are refractory to other chemotherapies as well as early line therapy for these genotypes.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Bovine Foamy Virus Transactivator (BTas) Interacts with Cellular RelB to Enhance Viral Transcription. 20844054

    Viruses are obligate intracellular parasites that depend on cellular machinery for their efficient transcription and replication. In a previous study we reported that bovine foamy virus (BFV) is able to activate the nuclear factor (NF)-κB pathway through the action of its transactivator (BTas) to enhance viral transcription. However, the mechanism used by NF-κB to enhance BFV transcription remains elusive. To address this question we employed a yeast two-hybrid assay to screen for BTas-interacting proteins. We found that RelB, a member of NF-κB protein family, interacts with BTas. We confirmed the putative RelB-BTas interaction in vitro and in vivo, and identified the protein regions responsible for the RelB-BTas interaction. Using a luciferase reporter assay, we next showed that RelB enhances BFV transcription (BTas-induced LTR transactivation), and that this process requires both localization of the RelB-BTas interaction in the nucleus and the Rel homology domain of RelB. Knockdown of cellular endogenous RelB protein using siRNA significantly attenuated BTas-induced LTR transcription. The results of ChIP analysis showed that endogenous RelB binds to the viral LTR in BFV-infected cells. Together, these results suggest that BFV engages RelB protein as a cotransactivator of BTas to enhance viral transcription. In addition, our findings indicate that BFV infection up-regulates cellular RelB expression through BTas-induced NF-κB activation. Thus, this study demonstrates the existence of a positive feed-back circuit in which BFV utilizes the host's NF-κB pathway through RelB protein for efficient viral transcription.
    Document Type:
    Reference
    Product Catalog Number:
    17-295
    Product Catalog Name:
    Chromatin Immunoprecipitation (ChIP) Assay Kit

  • Expression of adenosine receptors in the preglomerular microcirculation. 12060585

    The purpose of this study was to systematically investigate the abundance of each of the adenosine receptor subtypes in the preglomerular microcirculation vs. other vascular segments and vs. the renal cortex and medulla. Rat preglomerular microvessels (PGMVs) were isolated by iron oxide loading followed by magnetic separation. For comparison, mesenteric microvessels, segments of the aorta (thoracic, middle abdominal, and lower abdominal), renal cortex, and renal medulla were obtained by dissection. Adenosine receptor protein and mRNA expression were examined by Western blotting, Northern blotting, and RT-PCR. Our results indicate that compared with other vascular segments and renal tissues, A1 and A2B receptor protein and mRNA are abundantly expressed in the preglomerular microcirculation, whereas A2A and A3 receptor protein and mRNA are barely detectable or undetectable in PGMVs. We conclude that, relative to other vascular and renal tissues, A1 and A2B receptors are well expressed in PGMVs, whereas A2A and A3 receptors are notably deficient. Thus A1 and A2B receptors, but not A2A or A3 receptors, may importantly regulate the preglomerular microcirculation.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Epigenetic regulation of the stem cell mitogen Fgf-2 by Mbd1 in adult neural stem/progenitor cells. 18689796

    Whether and how mechanisms intrinsic to stem cells modulate their proliferation and differentiation are two central questions in stem cell biology. Although exogenous basic fibroblast growth factor 2 (FGF-2/Fgf-2) is commonly used to expand adult neural stem/progenitor cells (NSPCs) in vitro, we do not yet understand the functional significance or the molecular regulation of Fgf-2 expressed endogenously by adult NSPCs. We previously demonstrated that methylated CpG binding protein 1 (MBD1/Mbd1) is a transcriptional repressor of Fgf-2 and is enriched in adult brains. Mbd1 deficiency in mice selectively affected adult neurogenesis and the differentiation of NSPCs. Here we show that an Mbd1 and DNA methylation-mediated epigenetic mechanism regulated the expression of stem cell mitogen Fgf-2 in adult NSPCs. Mbd1 bound to the Fgf-2 promoter and regulates its expression in adult NSPCs. In the absence of functional Mbd1, the Fgf-2 promoter was hypomethylated, and treatment with a DNA methylation inhibitor resulted in increased Fgf-2 expression in adult NSPCs. We further demonstrated that both acute knockdown of Mbd1 or overexpression of Fgf-2 in adult NSPCs inhibited their neuronal differentiation, which could be responsible for the neurogenic deficits observed in Mbd1-deficient mice. These data indicate that intrinsic epigenetic mechanisms play critical roles in the regulation of adult NSPC functions.
    Document Type:
    Reference
    Product Catalog Number:
    17-295
    Product Catalog Name:
    Chromatin Immunoprecipitation (ChIP) Assay Kit

  • Inhibition of HIV-1 Tat-mediated transcription by a coumarin derivative, BPRHIV001, through the Akt pathway. 21697490

    The human immunodeficiency virus type 1 (HIV-1)-encoded RNA-binding protein Tat is known to play an essential role in viral gene expression. In the search for novel compounds to inhibit Tat transactivity, one coumarin derivative, BPRHIV001, was identified, with a 50% effective concentration (EC(50)) against HIV-1 at 1.3 nM. BPRHIV001 is likely to exert its effects at the stage after initiation of RNAPII elongation since Tat protein expression and the assembly of the Tat/P-TEFb complex remained unchanged. Next, a reduction of the p300 protein level, known to modulate Tat function through acetylation, was observed upon BPRHIV001 treatment, while the p300 mRNA level was unaffected. A concordant reduction of phosphorylated Akt, which was shown to be closely related to p300 stability, was observed in the presence of BPRHIV001 and was accompanied by a decrease of phosphorylated PDPK1, a well-known Akt activator. Furthermore, the docking analysis revealed that the reduced PDPK1 phosphorylation likely resulted from the allosteric effect of interaction between BPRHIV001 and PDPK1. With strong synergistic effects with current reverse transcriptase inhibitors, BPRHIV001 has the potential to become a promising lead compound for the development of a novel therapeutic agent against HIV-1 infection.
    Document Type:
    Reference
    Product Catalog Number:
    MAB424
    Product Catalog Name:
    Anti-PCNA Antibody, clone PC10

  • Differential traffic of proximal tubule Na+ transporters during hypertension or PTH: NHE3 to base of microvilli vs. NaPi2 to endosomes. 15265767

    We previously reported that Na(+)/H(+) exchanger type 3 (NHE3) and NaPi2 are acutely retracted from the proximal tubule (PT) microvilli (MV) during acute hypertension [high blood pressure (BP)] or parathyroid hormone (PTH) treatment. By subcellular membrane fractionation, NHE3 and NaPi2 show indistinguishable redistribution patterns out of light-density into heavy-density membranes in response to either treatment consistent with a retraction from the apical MV to the intermicrovillar cleft region. This study aimed to examine the redistribution of PT NHE3 vs. NaPi2 by confocal and electron microscopy during high BP and during PTH treatment to determine whether their respective destinations overlap or are distinct. High-BP protocol: systolic BP was increased 50-60 mmHg by increasing peripheral resistance for 20 min; PTH protocol: rats were infused with 6.6 microg/kg iv of PTH followed by 0.1 microg.kg(-1).min(-1) infusion for 1 h. For light microscopy, rats were infused with 25 mg of horseradish peroxidase (HRP) 10 min before kidney fixation. Kidney slices were dual labeled with either NHE3 or NaPi2 and either clathrin-coated vesicle adaptor protein AP2 or endosome marker HRP. The results demonstrate retraction of NHE3 from the MV to the base of MV during either high-BP or PTH treatment: NHE3 staining did not retract below the AP2-stained domain or to HRP-labeled endosomes in either model. In comparison, NaPi2 was retracted from MV to below the AP2-stained region in both models, a little colocalizing with HRP staining. At the electron microscopic level with immunogold labeling, during high BP NHE3 was concentrated in a distinct domain in the base of the MV while NaPi2 moved to endosomes. The results demonstrate that there are divergent routes of retraction of PT NHE3 and NaPi2 from the MV during acute hypertension or PTH treatment: NHE3 is not internalized but remains at the base of the MV while NaPi2 is internalized.
    Document Type:
    Reference
    Product Catalog Number:
    AB3085
    Product Catalog Name:
    Anti-Na+/H+ Exchanger-3 Antibody