scholarly journals Unexpected lack of specificity of a rabbit polyclonal TAP-L (ABCB9) antibody

F1000Research ◽  
2015 ◽  
Vol 4 ◽  
pp. 125 ◽  
Author(s):  
Peter van Endert ◽  
Myriam Lawand
Keyword(s):  
Molecular Weight ◽  
Bone Marrow ◽  
Antigen Processing ◽  
Antibody Specificity ◽  
Whole Cell ◽  
Cell Lysates ◽  
Rabbit Polyclonal Antibody ◽  
Specific Reactivity ◽  
Deficient Mice ◽  
Multiple Pcr

In this article, we describe the surprising non-specific reactivity in immunoblots of a rabbit polyclonal antibody (ref. Abcam 86222) expected to recognize the transporter associated with antigen processing like (TAP-L, ABCB9) protein. Although this antibody, according to company documentation, recognizes a band with the expected molecular weight of 84 kDa in HeLa, 293T and mouse NIH3T3 whole-cell lysates, we found that this band is also present in immunoblots of TAP-L deficient bone marrow-derived dendritic cell (BMDC) whole-cell lysates in three independent replicates. We performed extensive verification by multiple PCR tests to confirm the complete absence of the ABCB9 gene in our TAP-L deficient mice. We conclude that the antibody tested cross-reacts with an unidentified protein present in TAP-L knockout cells, which coincidentally runs at the same molecular weight as TAP-L. These findings underline the pitfalls of antibody specificity testing in the absence of cells lacking expression of the target protein.

P2Z purinoceptor-associated pores induced by extracellular ATP in macrophages and J774 cells

1997 ◽  
Vol 273 (6) ◽  
pp. C1793-C1800 ◽  
Author(s):  
Robson Coutinho-Silva ◽  
Pedro Muanis Persechini
Keyword(s):  
Molecular Weight ◽  
Bone Marrow ◽  
Patch Clamp ◽  
Plasma Membranes ◽  
Extracellular Atp ◽  
Low Molecular Weight ◽  
Whole Cell ◽  
Voltage Dependent ◽  
J774 Cells ◽  
Bone Marrow Derived Cells

Millimolar concentrations of extracellular ATP (ATPo) can induce the permeabilization of plasma membranes of macrophages and other bone marrow-derived cells to low-molecular-weight solutes, a phenomenon that is the hallmark of P2Z purinoceptors. However, patch-clamp and whole cell electrophysiological experiments have so far failed to demonstrate the existence of any ATPo-induced P2Z-associated pores underlying this permeabilization phenomenon. Here, we describe ATPo-induced pores of 409 ± 33 pS recorded using cell-attached patch-clamp experiments performed in macrophages and J774 cells. These pores are voltage dependent and display several properties of the P2Z-associated permeabilization phenomenon: they are permeable to both large cations and anions, such as tris(hydroxymethyl)aminomethane, N-methyl-d-glucamine, and glutamate; their opening is favored at temperatures higher than 30°C; they are blocked by oxidized ATP and Mg2+; and they can be triggered by 3′- O-(4-benzoylbenzoyl)-ATP but not by UTP or ADP. We conclude that the pores described in this report are associated with the P2Z permeabilization phenomenon.

Aberrantly Expressed Neutrophil Elastase (ELA2) Cleaves Cyclin E (CCNE) in the Nucleus and Cytoplasm of Acute Lymphocytic Leukemia Yielding Novel Leukemia-Associated Antigens.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4429-4429
Author(s):  
Ken Ishiyama ◽  
Yukio Kondo ◽  
Eric D. Wieder ◽  
Sijie Lu ◽  
Jeffrey J. Molldrem
Keyword(s):  
Bone Marrow ◽  
Cell Division ◽  
Bone Marrow Cells ◽  
Lymphocytic Leukemia ◽  
Leukemia Cells ◽  
Fusion Product ◽  
Leukocyte Elastase ◽  
Elastase Inhibitor ◽  
Whole Cell ◽  
Cell Lysates

Abstract We have shown that donor-derived cytotoxic T lymphocytes (CTL) are specific for two HLA-A2-restricted peptides derived from CCNE (CCNE1144–152, ILLDWLMEV and CCNE2144–152, ILLDWLLEV) specifically lyse lymphoid and myeloid leukemia cells that aberrantly express CCNE. The CCNE protein is overexpressed in AML, ALL, and CML, and in solid tumors such as breast, lung, and gastric cancers. Furthermore, five low molecular weight forms (LMWFs) of CCNE, which are constitutively active to promote cell division, are found only in malignant cells, though it is not known how LMWFs are formed in leukemia. We hypothesized that the cleavage of CCNE into LMWFs occurs by enzymatic cleavage from aberrantly expressed ELA2 in leukemia, which renders the leukemia susceptible to killing by ELA2- and CCNE-specific CTL. Whole cell lysates from U937, HL60, and bone marrow from patients with B-ALL, but not from PBMC or bone marrow cells from healthy donors or ALL patients in remission, showed high expression of CCNE and LMWF by western blot (WB). Recombinant ELA2 added to B cell-derived whole cell lysates increased LMWFs, and the leukocyte elastase inhibitor Elafin prevented this cleavage. Subcellular fractions studied by coimmunoprecipitation showed that ELA2 was bound to CCNE in the nucleus, cytoplasm, and membrane-bound organelles of B-ALL, but not in normal cells. Because nuclear expression of CCNE increases during normal cell division, we studied healthy donor PBMC stimulated with anti-CD3 and anti-CD28 and found no expression of ELA2 or CCNE LMWFs by WB. We conclude that ELA2, normally expressed only in myeloid cells, is also expressed in some ALL blasts, and this data explains how CCNE LMWFs are formed in leukemia. Our findings also suggest how ELA2-mediated cleavage of the PML-RARα fusion product, required for leukemic transformation, could occur when ELA2 is aberrantly expressed in the nucleus. Finally, this work implies that overexpression of CCNE and the LMWFs that contain the immunogenic peptides could increase susceptibility of leukemia cells to CCNE-CTL lysis.

scholarly journals The Proteasome Immunosubunit Multicatalytic Endopeptidase Complex-Like 1 Is a T-Cell-Intrinsic Factor Influencing Homeostatic Expansion

2007 ◽  
Vol 76 (3) ◽  
pp. 1207-1213 ◽  
Author(s):  
Dietmar M. W. Zaiss ◽  
Natascha de Graaf ◽  
Alice J. A. M. Sijts
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
Cell Population ◽  
Antigen Processing ◽  
Cd8 T Cell ◽  
Lymphoid Organs ◽  
T Cell Subsets ◽  
Class I Mhc ◽  
Mhc I ◽  
Deficient Mice

ABSTRACT Homeostatic regulatory mechanisms maintain the constant ratios between different lymphocyte subsets in the secondary lymphoid organs. How this dynamic equilibrium is achieved, in particular following the clonal expansion and subsequent contraction of different cells after infection, remains poorly understood. Expression of the proteasome immunosubunits has been shown to influence not only major histocompatibility complex class I (MHC-I) antigen processing and thereby T-cell responses, but also the CD4/CD8 T-cell ratios in lymphoid organs. We examined the relationships between these different immunosubunit-mediated effects in mice of various proteasome subunit compositions during infection with Listeria monocytogenes. Mice that lacked the immunosubunit multicatalytic endopeptidase complex-like 1 (MECL-1) maintained enhanced CD4/CD8 T-cell ratios during infection, while MHC-I surface levels resembled those in wild-type (wt) mice. LMP7 gene-deficient mice, on the other hand, showed reduced MHC-I expression, while their splenic CD4/CD8 ratios were similar to those in wt mice. Remarkably, analysis of bone marrow-chimeric immunosubunit gene-deficient mice, reconstituted with a mixture of wt and LMP7- plus MECL-1-deficient bone marrow, revealed that the LMP7- plus MECL-1-deficient T-cell population maintained a higher CD4/CD8 T-cell ratio than the wt T-cell population before, during, and after infection and T-cell memory formation. Since in these mice the immunosubunit-positive and immunosubunit-negative T-cell populations were selected in the same thymus and expanded in the same lymphoid environments, our findings indicate that MECL-1 influences the homeostatic equilibrium between T-cell subsets, not through indirect extracellular signals, such as MHC-I expression or the cytokine milieu, but through direct effects on T-cell-intrinsic processes.

scholarly journals Development and Characterization of Two Types of Surface Displayed Levansucrases for Levan Biosynthesis

Catalysts ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 757
Author(s):  
Huiyi Shang ◽  
Danni Yang ◽  
Dairong Qiao ◽  
Hui Xu ◽  
Yi Cao
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Weight ◽  
Large Scale ◽  
Surface Display ◽  
Yeast Surface Display ◽  
Fusion Partner ◽  
Whole Cell ◽  
Food Industries ◽  
Yeast Surface ◽  
The Stability

Levan has wide applications in chemical, cosmetic, pharmaceutical and food industries. The free levansucrase is usually used in the biosynthesis of levan, but the poor reusability and low stability of free levansucrase have limited its large-scale use. To address this problem, the surface-displayed levansucrase in Saccharomyces cerevisiae were generated and evaluated in this study. The levansucrase from Zymomonas mobilis was displayed on the cell surface of Saccharomyces cerevisiae EBY100 using a various yeast surface display platform. The N-terminal fusion partner is based on a-agglutinin, and the C-terminal one is Flo1p. The yield of levan produced by these two whole-cell biocatalysts reaches 26 g/L and 34 g/L in 24 h, respectively. Meanwhile, the stability of the surface-displayed levansucrases is significantly enhanced. After six reuses, these two biocatalysts retained over 50% and 60% of their initial activities, respectively. Furthermore, the molecular weight and polydispersity test of the products suggested that the whole-cell biocatalyst of levansucrase displayed by Flo1p has more potentials in the production of levan with low molecular weight which is critical in certain applications. In conclusion, our method not only enable the possibility to reuse the enzyme, but also improves the stability of the enzyme.

scholarly journals Bone Marrow Transplantation in Apolipoprotein E–Deficient Mice

1997 ◽  
Vol 17 (11) ◽  
pp. 3117-3126 ◽  
Author(s):  
Miranda Van Eck ◽  
Nicole Herijgers ◽  
John Yates ◽  
Nigel J. Pearce ◽  
Peter M. Hoogerbrugge ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Transplantation ◽  
Apolipoprotein E ◽  
Marrow Transplantation ◽  
Deficient Mice

scholarly journals Tyro3/Axl/Mertk-deficient mice develop bone marrow edema which is an early pathological marker in rheumatoid arthritis

PLoS ONE ◽  
2018 ◽  
Vol 13 (10) ◽  
pp. e0205902 ◽  
Author(s):  
Claire E. J. Waterborg ◽  
Marije I. Koenders ◽  
Peter L. E. M. van Lent ◽  
Peter M. van der Kraan ◽  
Fons A. J. van de Loo
Keyword(s):  
Rheumatoid Arthritis ◽  
Bone Marrow ◽  
Bone Marrow Edema ◽  
Deficient Mice ◽  
Marrow Edema

scholarly journals TP53INP1 deficiency maintains murine B lymphopoiesis in aged bone marrow through redox-controlled IL-7R/STAT5 signaling

2018 ◽  
Vol 116 (1) ◽  
pp. 211-216 ◽  
Author(s):  
Bochra Zidi ◽  
Christelle Vincent-Fabert ◽  
Laurent Pouyet ◽  
Marion Seillier ◽  
Amelle Vandevelde ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Immune Cell ◽  
B Lymphopoiesis ◽  
Hematopoietic Stem ◽  
Chronic Oxidative Stress ◽  
The Impact ◽  
Deficient Mice

Bone marrow (BM) produces all blood and immune cells deriving from hematopoietic stem cells (HSCs). The decrease of immune cell production during aging is one of the features of immunosenescence. The impact of redox dysregulation in BM aging is still poorly understood. Here we use TP53INP1-deficient (KO) mice endowed with chronic oxidative stress to assess the influence of aging-associated redox alterations in BM homeostasis. We show that TP53INP1 deletion has no impact on aging-related accumulation of HSCs. In contrast, the aging-related contraction of the lymphoid compartment is mitigated in TP53INP1 KO mice. B cells that accumulate in old KO BM are differentiating cells that can mature into functional B cells. Importantly, this phenotype results from B cell-intrinsic events associated with defective redox control. Finally, we show that oxidative stress in aged TP53INP1-deficient mice maintains STAT5 expression and activation in early B cells, driving high Pax5 expression, which provides a molecular mechanism for maintenance of B cell development upon aging.

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