SARS-CoV-2 antibodies seroprevalence in dogs from France using ELISA and an automated western blotting assay

Background/Aims: Oxygen glucose deprivation (OGD)/re-oxygenation (OGDR) exposure to myocardial cells mimics ischemia-reperfusion injuries. We studied the potential activity of ciliary neurotrophic factor (CNTF) on OGDR-treated myocardial cells. Methods: CNTF and CNTFR expression were tested by RT-PCR assay and Western blotting assay. Cell viability and death were tested by MTT assay and LDH release assay, respectively. Akt-Nrf2 signalings were tested by Western blotting assay and qPCR assay. Results: CNTF and its receptor CNTFR were functionally expressed in established H9c2 myocardial cells and primary murine myocardiocytes. Pretreatment of CNTF significantly attenuated OGDR-induced viability reduction and death in myocardial cells. Further studies show that in the myocardial cells CNTF activated NF-E2-related factor 2 (Nrf2) signaling to inhibit OGDR-induced reactive oxygen species (ROS) production and programmed necrosis, preventing adenine nucleotide translocator 1 (ANT-1)-p53-cyclophilin D (Cyp-D) mitochondrial association and mitochondrial depolarization. Nrf2 silencing or knockout almost abolished CNTF-induced H9c2 cytoprotection against OGDR. CNTF activated Akt in H9c2 cells and primary murine myocardiocytes. Conversely, Akt blockage by the pharmacological inhibitors not only blocked CNTF-induced Nrf2 Ser-40 phosphorylation and activation, but also nullified anti-OGDR actions by CNTF in myocardial cells. Conclusion: CNTF activates Akt-Nrf2 signaling to protect myocardial cells from OGDR.

Download Full-text

Western Blotting Assay for Egr-1 Immediate Early Gene in Brain Tissue of Zebrafish Applied to Neuroethological Study

Neuromethods - Zebrafish Protocols for Neurobehavioral Research ◽

10.1007/978-1-61779-597-8_26 ◽

2012 ◽

pp. 331-342

Author(s):

Augusto Barbosa ◽

Anette Hoffmann ◽

Fabiana Luca Alves ◽

Carla Patricia Bejo Wolkers ◽

Fernando Massaru Hoshiko ◽

...

Keyword(s):

Brain Tissue ◽

Western Blotting ◽

Immediate Early Gene ◽

Early Gene ◽

Immediate Early ◽

Western Blotting Assay

Download Full-text

Bimodal size distribution immuno-quantum dots for fluorescent western blotting assay with high sensitivity and extended dynamic range

Microchimica Acta ◽

10.1007/s00604-020-04578-z ◽

2020 ◽

Vol 187 (11) ◽

Author(s):

Wannian Yan ◽

Lingzhi Fan ◽

Jin Li ◽

Yijiang Wang ◽

Huanxing Han ◽

...

Keyword(s):

Quantum Dots ◽

Size Distribution ◽

Western Blotting ◽

Dynamic Range ◽

High Sensitivity ◽

Bimodal Size Distribution ◽

Western Blotting Assay ◽

Extended Dynamic

Download Full-text

Bovine milk-derived exosomes mediated delivery of resibufogenin and its effect on platinum-resistant ovarian carcinoma.

Journal of Clinical Oncology ◽

10.1200/jco.2019.37.15_suppl.e17065 ◽

2019 ◽

Vol 37 (15_suppl) ◽

pp. e17065-e17065 ◽

Cited By ~ 1

Author(s):

Guannan Zhou ◽

Jingxin Ding

Keyword(s):

Actin Cytoskeleton ◽

Ovarian Carcinoma ◽

Western Blotting ◽

Bovine Milk ◽

Qpcr Assay ◽

Mrna Sequencing ◽

Platinum Resistant ◽

Western Blotting Assay ◽

Regulation Of Actin Cytoskeleton

e17065 Background: Platinum-resistant ovarian carcinoma presents poor prognosis owing to its chemotherapeutic resistance. Resibufogenin is a kind of traditional Chinese medicine with potential anti-cancer ability. Exosomes are naturally occurring membrane particles that mediate intercellular communication by delivering molecular information between cells. The purpose of the current study was to evaluate the effect of bovine-milk-derived exosomes as carriers of resibufogenin to Platinum-resistant ovarian carcinoma and investigate the mechanism. Methods: Exosomes were isolated from bovine-milk using differential centrifugation and characterized by electron microscopy, nanoparticle tracking analysis, and western blot. Drug screening among 172 traditional Chinese medicines to find out resibufogenin. The exosomes were loaded with resibufogenin and the effectiveness of exosome-mediated drug was assessed with viability assay, transwell assay and flow cytometry assay. The distribution of exosome-delivered resibufogenin in cells was inspected by confocal microscopy. Xenograft mouse models were constructed to clarify the treatment response in vivo. mRNA sequencing assay, qPCR assay, immunohistochemistry assay and western blotting assay were used to detect and verify the targeted pathways. Results: Our main finding that resibufogenin inhibits the proliferation, migration and invasion in platinum-resistant ovarian carcinoma. Bovine-milk-derived exosomes delivered resibufogenin into platinum-resistant ovarian carcinoma and enhanced the cytotoxicity of resibufogenin. In vivo experiment demonstrated that resibufogenin suppressed the growth of xenograft tumors, with lower ki-67 expression and higher tunel expression. The mRNA sequencing assay, qPCR assay, immunohistochemistry assay and western blotting assay showed that the PI3K/AKT and Regulation of actin cytoskeleton signaling pathways account for the results above. Conclusions: Bovine-milk-derived exosomes can be used as effective carriers of resibufogenin to platinum-resistant ovarian carcinoma. Bovine-milk-derived exosomes-loading resibufogenin inhibits platinum-resistant ovarian carcinoma growth and migration in vitro and in vivo through PI3K/AKT and Regulation of actin cytoskeleton signaling pathway.

Download Full-text

Circular RNA circASPM Promotes the Progression of Glioblastoma by Acting as a Competing Endogenous RNA to Regulate miR-130b–3p/E2F1 Axis

10.21203/rs.3.rs-132678/v1 ◽

2020 ◽

Author(s):

Dianqi Hou ◽

Zhenlin Wang ◽

Haimeng Li ◽

Juan Liu ◽

Yaohua Liu ◽

...

Keyword(s):

Western Blotting ◽

Circular Rna ◽

Luciferase Reporter ◽

Circular Rnas ◽

Primary Malignancy ◽

Rna Immunoprecipitation ◽

Western Blotting Assay

Abstract background: Glioblastoma Multiform (GBM) is the primary malignancy with the highest incidence and worst prognosis in the adult CNS. Circular RNAs (circRNAs) are a novel and widely diverse class of endogenous non-coding RNAs that can promote or inhibit gliomagenesis. Our study aimed to explore the role of circASPM in GBM and its molecular mechanism.Methods: Levels of circASPM, miR-130b-3p and E2F1 were determined by quantitative real-time PCR (qRT-PCR) or western blotting assay. MTS, Edu, neurospheres formation and extreme limiting dilution assays were used to detect the tumorigenesis and proliferation of GSCs in vitro. The interactions between miR-130b-3p and circASPM or E2F1 was demonstrated via qPCR, western blotting, dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. Xenograft experiments was used to analyze tumor growth in vivo.Results: CircASPM was overexpressed in GBM and could promote the tumorigenesis and proliferation of GSCs both in vitro and in vivo. Mechanistically, circASPM up-regulated the expression of E2F1 in GSCs via miR-130b-3p sponging. We furtherly demonstrated that circAPSM could promote the GSCs proliferation via E2F1 up-regulating. Therefore, our study identified a novel circRNA and its possible mechanism in the development and tumorigenesis of GBM.Conclusions: CircASPM can promote GBM progression via regulating miR-130b-3p/E2F1 axis, suggesting that circAPSM could provide an effective biomarker for GBM diagnosis and prognostic evaluation and possibly being used for molecular targeted therapy.

Download Full-text

Hydrogen Enhanced the Myogenic Differentiation of Adipose Mesenchymal Stem Cells Through P38 MAPK Signaling Pathway

10.21203/rs.3.rs-1042384/v1 ◽

2021 ◽

Author(s):

Wenyong Fei ◽

Erkai Pang ◽

Lei Hou ◽

Jihang Dai ◽

Mingsheng Liu ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

P38 Mapk ◽

Western Blotting ◽

Myogenic Differentiation ◽

Mapk Signaling ◽

Blue Staining ◽

Adipose Mesenchymal Stem Cells ◽

Western Blotting Assay

Abstract Purpose: This study aims to clarify the systems underlying regulation and regulatory roles of hydrogen in the myogenic differentiation of adipose mesenchymal stem cells (ADSCs). Materials and methods: In this study, ADSCs acted as an in vitro myogenic differentiating mode. First, the Alamar blue Staining and mitochondrial tracer technique were used to verify whether hydrogen could promote cell proliferation. In addition, this study assessed myogenic differentiating markers (e.g., Myogenin, Mhc and Myod protein expressions) based on the Western blotting assay, analysis on cellular morphological characteristics (e.g., Myotube number, length, diameter and maturation index), RT-PCR (Mhc and Myod mRNA expression) and Immunofluorescence analysis (Desmin, Myosin and β-actin protein expression). Lastly, to verify the myogenic differentiating system of hydrogen, Western blotting assay was performed to detect p38 and p-p38 proteins expressions. Results: Hydrogen can remarkably enhance the proliferation of ADSCs in vitro by increasing the number of single-cell mitochondria and by up-regulating the expression of myogenic biomarkers (e.g., Myod, Mhc and myotube formation). The expressions of both p38 and p-p38 were up-regulated by hydrogen. The differentiating ability was suppressed when the cells were cultivated in combination with SB203580 (p38 MAPK signal pathway inhibitor). Conclusions: The present study initially indicated that hydrogen can promote myogenic differentiation via the p38 MAPK pathway. Thus, the mentioned results present insights into myogenic differentiation and are likely to generate one potential alternative strategy for skeletal muscle related diseases.

Download Full-text

Western blotting assay of transketolase concentration in human hemolysates

Analytical Biochemistry ◽

10.1016/0003-2697(88)90345-4 ◽

1988 ◽

Vol 168 (2) ◽

pp. 470-475 ◽

Cited By ~ 4

Author(s):

Tohoru Takeuchi ◽

Eun Hee Jung ◽

Kohsuke Nishino ◽

Yoshinori Itokawa

Keyword(s):

Western Blotting ◽

Western Blotting Assay

Download Full-text

Ripasudil alleviated the inflammation of RPE cells by targeting the miR-136-5p/ROCK/NLRP3 pathway

10.21203/rs.2.20697/v1 ◽

2020 ◽

Author(s):

Zhao Gao ◽

Qiang Li ◽

Yunda Zhang ◽

Haiyan Li ◽

Xiaohong Gao ◽

...

Keyword(s):

Western Blotting ◽

Inflammatory Diseases ◽

Normal Function ◽

Eye Diseases ◽

Luciferase Reporter ◽

Rpe Cells ◽

Reporter Assays ◽

Related Proteins ◽

Western Blotting Assay ◽

Stimulation Of

Abstract Introduction Inflammation of RPE cells lead to different kinds of eye diseases and affect the normal function of the retina. Furthermore, higher levels of ROCK1 and ROCK2 induced injury of endothelial cells and many inflammatory diseases of the eyes. Ripasudil which was used for the treatment of glaucoma was one kind of the inhibitor of ROCK1 and ROCK2. But whether the ripasudil could relieved the LPS induced inflammation damage of RPE cells was not clear.Material and methods We used the LPS to stimulate ARPE-19 cells which is the RPE cell line. After that, we detected the levels of ROCK1 and ROCK2 by western-blotting assay after the stimulation of LPS and treatment of ripasudil. Then luciferase reporter assays were used to confirm the targeted effect of miR-136-5p on ROCK1 and ROCK2. At last, the levels of NLRP3, ASC, caspase1, IL-1β and IL-18 were detected with the western-blotting after the knockdown of miR-136-5p.Results The levels of ROCK1, ROCK2 and miR-136-5p in ARPE-19 cells was promoted after the stimulation of LPS. After the treatment of ripasudil, the ROCK1, ROCK2 and miR-136-5p was suppressed. The miR-136-5p targeted and inhibited the expression of ROCK1 and ROCK2. Inflammation related proteins NLRP3, ASC, caspase1, IL-1β and IL-18 was inhibited after the treatment of ripasudil. However, the expression of these proteins was rescued after the knockdown of the miR-136-5p.Conclusion Ripasudil relieved the inflammatory injury of RPE cells by upregulating miR-136-5p and therefore inhibiting the expression of ROCK1, ROCK2, NLRP3, ASC, caspase1, IL-1β and IL-18.

Download Full-text

Akt Inhibition Improves the Efficacy of Cabazitaxel Nanomedicine in Preclinical Taxane-Resistant Cancer Models

10.21203/rs.3.rs-117705/v1 ◽

2020 ◽

Author(s):

Tongyu Li ◽

Linlin Shi ◽

Jianqin Wan ◽

Xiaoxiao Hu ◽

Wanzhi Chen ◽

...

Keyword(s):

Drug Resistance ◽

Western Blotting ◽

Combined Treatment ◽

Xenograft Model ◽

Akt Inhibitor ◽

Defense Proteins ◽

Western Blotting Assay ◽

Resistant Cells

Abstract Background: Drug resistance continues to be a major clinical challenge in achieving cures in cancer patients. Cabazitaxel has shown the ability to surmount drug resistance through bypassing the transporter-mediated drug expulsion; however, the substantially high toxicity in patients hampered its clinical application. In addition, upregulation of certain self-defense proteins (e.g, Akt) was reportedly involved in drug resistance, which may further compromise the activity of cabazitaxel. We have previously developed several prodrug-based strategies to deliver nanoparticles encapsulating cabazitaxel derivatives in tumors with enhanced efficacy and improved in vivo tolerability. Therefore, we hypothesized that combing cabazitaxel nanotherapeutics with a pan-Akt inhibitor MK-2206 would synergistically eliminate the resistant cancers with reduced systemic toxicity.Methods: Activation of Akt in resistant cancers upon cabazitaxel treatment was determined by western blotting assay. The effect of combing MK-2206 with cabazitaxel on cell viability was evaluated by CCK-8 assay. To improve the in vivo biocompatibility, the delivery of potent cabazitaxel was feasibly achieved via the integration of oligolactide-conjugated cabazitaxel into the PEG-b-PLA matrix. Both the synergism and safety of cabazitaxel nanomedicine-based combination were evaluated through a series of in vitro and in vivo experiments, including Western blotting assay, CCK-8 assay, EdU assay, flow cytometry, migration assay, transwell assay, MTD study, myelosuppression study, nude mouse xenograft, and immunostaining analyses. Results: We found that resistant cells adapted to activate Akt signaling upon cabazitaxel treatment, which potentially discounts the efficacy of cabazitaxel. The addition of MK-2206 reversed this situation and potentiated the activity of cabazitaxel nanomedicine against resistant cells. Mechanistically, suppression of the Akt pathway increased the microtubule-stabilizing effect of cabazitaxel. Their collaboration was demonstrated to maximize the efficacy in a xenograft model bearing paclitaxel-resistant tumors. In particular, the nanoformulation substantially improved drug tolerability in animals, and combined treatment with MK-2206 was proven to be safe for synergistic cancer therapy. Conclusion: The preclinical studies demonstrate the therapeutic efficacy of our binary system consisting of a better-tolerated nanotherapy and a specific pathway modulator against resistant cells, thereby highlighting the potential applications for the clinical treatment of patients with multidrug-resistant malignancies.

Download Full-text