![]() ![]() Drp1 is primarily located in the cytosol. Dynamin-related protein 1 (Drp1) is a central regulator in mitochondrial fission. Mitochondrial morphology is dynamically controlled by continuous fission and fusion. Alterations in mitochondrial morphology increases the susceptibility of the heart to ischemia/reperfusion injury 4, 5, indicating the important role of mitochondrial integrity in the protection of cardiac function. Although inflammation, oxidative stress and endoplasmic reticulum stress are involved in cardiac injury, accumulating evidence demonstrates that mitochondrial dysfunction is an initial cause for these events 2, 3. In cardiomyocytes, mitochondrial homeostasis plays a key role in maintaining heart function in response to metabolic stress 1. These findings suggest that inhibition of extracellular succinate-mediated GPR91 activation might be a potential therapeutic strategy for protecting cardiomyocytes from ischemic injury. Taken together, these results showed that in response to cardiac ischemia, succinate release activated GPR91 and induced mitochondrial fission via regulation of PKCδ and ERK1/2 signaling branches. We further showed that inhibition of succinate release and GPR91 signaling ameliorated oxygen–glucose deprivation-induced injury in cardiomyocytes and isoproterenol-induced myocardial ischemia injury in mice. As a result, enhanced localization of MFF and Drp1 in mitochondria promoted mitochondrial fission, leading to mitochondrial dysfunction and cardiomyocyte apoptosis. The results showed that extracellular succinate promoted the translocation of dynamin-related protein 1 (Drp1) to mitochondria via protein kinase Cδ (PKCδ) activation, and induced mitochondrial fission factor (MFF) phosphorylation via extracellular signal-regulated kinases-1/2 (ERK1/2) activation in a GPR91-dependent manner. This work aims to investigate whether or not extracellular succinate accumulation and its targeted G protein-coupled receptor-91 (GPR91) activation induce cardiac injury through mitochondrial impairment. Figure 1 shows that EGF-induced phosphorylation of EGFR and MEK1 was observed in control cell lines (lanes 1–4) and not in the EGFR knockout cell lines (lanes 5 and 6).Altered mitochondrial metabolism acts as an initial cause for cardiovascular diseases and metabolic intermediate succinate emerges as a mediator of mitochondrial dysfunction. The blot was imaged using the Invitrogen iBright FL1000 Imaging System the signals were normalized using an antibody specific for the housekeeping protein glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Cell lysates were then prepared, separated by protein gel electrophoresis, transferred to a membrane, and probed using spectrally distinct fluorescent antibodies specific for EGFR and MEK1 and the corresponding phosphoproteins ( Table 1). EGFR knockout cells created using CRISPR-Cas9 gene editing technology were treated with epidermal growth factor (EGF). To demonstrate its utility, we used multiplex western blotting to illustrate the efficient knockout of the gene encoding the epidermal growth factor receptor (EGFR), and the effects of this knockout on the downstream mitogen-activated protein kinase 1 (MEK1) ( Figure 1). Furthermore, advances in western blot imaging equipment and software enable streamlined protein quantitation. In contrast, traditional chemiluminescent western blotting requires multiple rounds of probing, stripping, and reprobing to collect the same pool of data.īy minimizing the need to run multiple protein gels or reprobe blots, multiplex western blotting saves a significant amount of time, effort, and cost. Probing the western blot with several fluorescently labeled antibodies that emit light at different wavelengths enables the simultaneous detection of multiple protein targets on a single blot. For example, antibody pairs that recognize a specific protein and corresponding phosphoprotein can be used to determine the phosphorylation state of a putative downstream protein in a signaling pathway. The effect of gene knockouts on individual proteins in a signaling pathway can be determined using western blotting with pathway-specific antibodies. Analyze gene knockouts with multiplex western blotting ![]()
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |