RB459 and RB462 antibodies recognize mouse Pannexin1 protein by immunofluorescent staining

Rusiecka, Olga M.; Roth, Christel L; Kwak, Brenda R.; Molica, Filippo

doi:10.24450/journals/abrep.2019.e39

Cited by 4 publications

(6 citation statements)

References 5 publications

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“…EA.hy926 cells were stained with anti-Panx1 HRB462 mini-body (Geneva Antibody Facility) diluted at 1/250 as previously described . H9c2 cells were stained with custom-made anti-mPanx1 414–425 diluted at 1/500 using previously established protocols .…”

Section: Methodsmentioning

confidence: 99%

“…EA.hy926 cells were stained with anti-Panx1 HRB462 mini-body (Geneva Antibody Facility) diluted at 1/250 as previously described. 87 H9c2 cells were stained with custom-made anti-mPanx1 414–425 diluted at 1/500 using previously established protocols. 62 Briefly, cells were fixed with PFA (4%) for 15 min and permeabilized in Triton X-100 (0.3%; vol/vol) for 15 min.…”

Section: Methodsmentioning

confidence: 99%

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Structure-Based Design and Synthesis of Stapled ¹⁰Panx1 Analogues for Use in Cardiovascular Inflammatory Diseases

Lamouroux,

Tournier,

Iaculli

et al. 2023

J. Med. Chem.

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Following a rational design, a series of macrocyclic ("stapled") peptidomimetics of 10 Panx1, the most established peptide inhibitor of Pannexin1 (Panx1) channels, were developed and synthesized. Two macrocyclic analogues SBL-PX1-42 and SBL-PX1-44 outperformed the linear native peptide. During in vitro adenosine triphosphate (ATP) release and Yo-Pro-1 uptake assays in a Panx1expressing tumor cell line, both compounds were revealed to be promising bidirectional inhibitors of Panx1 channel function, able to induce a two-fold inhibition, as compared to the native 10 Panx1 sequence. The introduction of triazole-based cross-links within the peptide backbones increased helical content and enhanced in vitro proteolytic stability in human plasma (>30-fold longer half-lives, compared to 10 Panx1). In adhesion assays, a "double-stapled" peptide, SBL-PX1-206 inhibited ATP release from endothelial cells, thereby efficiently reducing THP-1 monocyte adhesion to a TNF-α-activated endothelial monolayer and making it a promising candidate for future in vivo investigations in animal models of cardiovascular inflammatory disease.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Structure-Based Design and Synthesis of Stapled ¹⁰Panx1 Analogues for Use in Cardiovascular Inflammatory Diseases

Lamouroux,

Tournier,

Iaculli

et al. 2023

J. Med. Chem.

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“…Immunostaining was performed on 7 μm-thick cryosections or cells using antibodies against Ly6G (1/50, BD Pharmingen), F4/80 (1/250, Proteintech) or Panx1 (1/250, Geneva Antibody Facility 25 or anti-mPanx1 414–425 , 1/500, Aves Labs. 26 ) Briefly, after 15 min fixation with 4% paraformaldehyde, heart sections were permeabilized for 15 min with 0.3% Triton X-100, incubated in 0.5 M NH 4 Cl and blocked in a PBS solution containing 2% BSA.…”

Section: Methodsmentioning

confidence: 99%

Mitochondrial pannexin1 controls cardiac sensitivity to ischaemia/reperfusion injury

Rusiecka,

Molica,

Nielsen

et al. 2023

Cardiovascular Research

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Aims No effective therapy is available in clinics to protect the heart from ischaemia/reperfusion (I/R) injury. Endothelial cells are activated after I/R, which may drive the inflammatory response by releasing ATP through pannexin1 (Panx1) channels. Here, we investigated the role of Panx1 in cardiac I/R. Methods and results Panx1 was found in cardiac endothelial cells, neutrophils, and cardiomyocytes. After in vivo I/R, serum Troponin-I, and infarct size were less pronounced in Panx1−/− mice, but leukocyte infiltration in the infarct area was similar between Panx1−/− and wild-type mice. Serum Troponin-I and infarct size were not different between mice with neutrophil-specific deletion of Panx1 and Panx1fl/fl mice, suggesting that cardioprotection by Panx1 deletion rather involved cardiomyocytes than the inflammatory response. Physiological cardiac function in wild-type and Panx1−/− hearts was similar. The time to onset of contracture and time to maximal contracture were delayed in Panx1−/− hearts, suggesting reduced sensitivity of these hearts to ischaemic injury. Moreover, Panx1−/− hearts showed better recovery of left ventricle developed pressure, cardiac contractility, and relaxation after I/R. Ischaemic preconditioning failed to confer further protection in Panx1−/− hearts. Panx1 was found in subsarcolemmal mitochondria (SSM). SSM in WT or Panx1−/− hearts showed no differences in morphology. The function of the mitochondrial permeability transition pore and production of reactive oxygen species in SSM was not affected, but mitochondrial respiration was reduced in Panx1−/− SSM. Finally, Panx1−/− cardiomyocytes had a decreased mitochondrial membrane potential and an increased mitochondrial ATP content. Conclusion Panx1−/− mice display decreased sensitivity to cardiac I/R injury, resulting in smaller infarcts and improved recovery of left ventricular function. This cardioprotective effect of Panx1 deletion seems to involve cardiac mitochondria rather than a reduced inflammatory response. Thus, Panx1 may represent a new target for controlling cardiac reperfusion damage.

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“…With the help of the Geneva Antibody Facility ( https://www.unige.ch/medecine/antibodies/ ), our group has recently raised a panel of mini-antibodies against a sequence (WRQAAFVDSY) in the EL1 of Panx1 ( Molica et al, 2019 ; Rusiecka et al, 2019 ). The antigen-binding fragment was fused to the human Fc ( Ahmad et al, 2012 ; Molica et al, 2019 ).…”

Section: Novel Classes Of Panx1 Channel Inhibitors: a Potential Appro...mentioning

confidence: 99%

“…The antigen-binding fragment was fused to the human Fc ( Ahmad et al, 2012 ; Molica et al, 2019 ). While some of the mini-antibodies appeared very useful for immunofluorescent microscopy, the anti-Panx1 HRB454 appeared to hold Panx1 channel inhibiting properties ( Molica et al, 2019 ; Rusiecka et al, 2019 ). In in vivo mouse models of venous and arterial thrombosis, HRB454 decreased hemostasis and thrombosis via inhibition of Panx1 channels ( Molica et al, 2019 ).…”

Section: Novel Classes Of Panx1 Channel Inhibitors: a Potential Appro...mentioning

confidence: 99%

Pannexin1 channels—a potential therapeutic target in inflammation

Rusiecka

Tournier

Molica

et al. 2022

Front. Cell Dev. Biol.

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An exaggerated inflammatory response is the hallmark of a plethora of disorders. ATP is a central signaling molecule that orchestrates the initiation and resolution of the inflammatory response by enhancing activation of the inflammasome, leukocyte recruitment and activation of T cells. ATP can be released from cells through pannexin (Panx) channels, a family of glycoproteins consisting of three members, Panx1, Panx2, and Panx3. Panx1 is ubiquitously expressed and forms heptameric channels in the plasma membrane mediating paracrine and autocrine signaling. Besides their involvement in the inflammatory response, Panx1 channels have been shown to contribute to different modes of cell death (i.e., pyroptosis, necrosis and apoptosis). Both genetic ablation and pharmacological inhibition of Panx1 channels decrease inflammation in vivo and contribute to a better outcome in several animal models of inflammatory disease involving various organs, including the brain, lung, kidney and heart. Up to date, several molecules have been identified to inhibit Panx1 channels, for instance probenecid (Pbn), mefloquine (Mfq), flufenamic acid (FFA), carbenoxolone (Cbx) or mimetic peptides like 10Panx1. Unfortunately, the vast majority of these compounds lack specificity and/or serum stability, which limits their application. The recent availability of detailed structural information on the Panx1 channel from cryo-electron microscopy studies may open up innovative approaches to acquire new classes of synthetic Panx1 channel blockers with high target specificity. Selective inhibition of Panx1 channels may not only limit acute inflammatory responses but may also prove useful in chronic inflammatory diseases, thereby improving human health. Here, we reviewed the current knowledge on the role of Panx1 in the initiation and resolution of the inflammatory response, we summarized the effects of Panx1 inhibition in inflammatory pathologies and recapitulate current Panx1 channel pharmacology with an outlook towards future approaches.

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RB459 and RB462 antibodies recognize mouse Pannexin1 protein by immunofluorescent staining

Abstract: The recombinant antibodies RB459 and RB462 recognize mouse Pannexin1 (Panx1) by immunofluorescent staining of a mouse cardiac endothelial cell line, a murine macrophage cell line and cryosections of mouse tissue.

Cited by 4 publications

References 5 publications

Structure-Based Design and Synthesis of Stapled ¹⁰Panx1 Analogues for Use in Cardiovascular Inflammatory Diseases

Structure-Based Design and Synthesis of Stapled ¹⁰Panx1 Analogues for Use in Cardiovascular Inflammatory Diseases

Mitochondrial pannexin1 controls cardiac sensitivity to ischaemia/reperfusion injury

Pannexin1 channels—a potential therapeutic target in inflammation

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RB459 and RB462 antibodies recognize mouse Pannexin1 protein by immunofluorescent staining

Abstract: The recombinant antibodies RB459 and RB462 recognize mouse Pannexin1 (Panx1) by immunofluorescent staining of a mouse cardiac endothelial cell line, a murine macrophage cell line and cryosections of mouse tissue.

Cited by 4 publications

References 5 publications

Structure-Based Design and Synthesis of Stapled 10Panx1 Analogues for Use in Cardiovascular Inflammatory Diseases

Structure-Based Design and Synthesis of Stapled 10Panx1 Analogues for Use in Cardiovascular Inflammatory Diseases

Mitochondrial pannexin1 controls cardiac sensitivity to ischaemia/reperfusion injury

Pannexin1 channels—a potential therapeutic target in inflammation

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Product

Resources

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Structure-Based Design and Synthesis of Stapled ¹⁰Panx1 Analogues for Use in Cardiovascular Inflammatory Diseases

Structure-Based Design and Synthesis of Stapled ¹⁰Panx1 Analogues for Use in Cardiovascular Inflammatory Diseases