Co-immunoprecipitation from Transfected Cells

2015 ◽  
pp. 381-389 ◽  
Author(s):  
Yoshinori Takahashi
Keyword(s):  
Transfected Cells

Dissection of the molecular mechanisms of protein targeting to the peroxisome using immunofluorescence and immunocryoelectron microscopies

1990 ◽  
Vol 48 (3) ◽  
pp. 44-45
Author(s):  
G-A. Keller ◽  
S. J. Gould ◽  
S. Subramani ◽  
S. Krisans
Keyword(s):  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Protein Targeting ◽  
Firefly Luciferase ◽  
Peroxisomal Enzyme ◽  
Transfected Cells ◽  
Peroxisomal Targeting ◽  
Targeting Signal ◽  
Series Of Experiments ◽  
Peroxisomal Targeting Signal

Subcellular compartments within eukaryotic cells must each be supplied with unique sets of proteins that must be directed to, and translocated across one or more membranes of the target organelles. This transport is mediated by cis- acting targeting signals present within the imported proteins. The following is a chronological account of a series of experiments designed and carried out in an effort to understand how proteins are targeted to the peroxisomal compartment.-We demonstrated by immunocryoelectron microscopy that the enzyme luciferase is a peroxisomal enzyme in the firefly lantern. -We expressed the cDNA encoding firefly luciferase in mammalian cells and demonstrated by immunofluorescence that the enzyme was transported into the peroxisomes of the transfected cells. -Using deletions, linker insertions, and gene fusion to identify regions of luciferase involved in its transport to the peroxisomes, we demonstrated that luciferase contains a peroxisomal targeting signal (PTS) within its COOH-terminal twelve amino acid.

scholarly journals Reduced Endothelin-2 and Hypoxic Signaling Pathways in Granulosa-Lutein Cells of PCOS Women

2021 ◽  
Vol 22 (15) ◽  
pp. 8216
Author(s):  
Magdalena Szymanska ◽  
Ketan Shrestha ◽  
Eliezer Girsh ◽  
Avi Harlev ◽  
Iris Eisenberg ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Target Genes ◽  
Hypoxic Response ◽  
Transfected Cells ◽  
Healthy Women ◽  
Periovulatory Follicles

Granulosa-lutein cells (GLCs) from PCOS women display reduced HIF-1α and EDN2 levels, suggesting their role in PCOS etiology. Here, we investigated the mechanisms involved in aberrant EDN2 expression in PCOS, and its association with HIF-1α. Various HIF-1α-dependent factors were studied in GLCs from PCOS and compared to normally ovulating women. MicroRNA-210 (miR-210), its target genes (SDHD and GPD1L), and HIF-1α-responsive genes (EDN2 and VEGFA) differed in GLCs from PCOS, compared with those of healthy women. Levels of miR-210—designated hypoxiamiR—and EDN2 were reduced in the PCOS GLCs; concomitantly, GPD1L and SDHD levels were elevated. Cultured GLCs retained low EDN2 expression and had low HIF-1α levels, providing evidence for a disrupted hypoxic response in the PCOS GLCs. However, VEGFA expression was elevated in these cells. Next, miR-210 levels were manipulated. miR-210-mimic stimulated EDN2 twice as much as the miR-NC-transfected cells, whereas miR-210-inhibitor diminished EDN2, emphasizing the importance of hypoxiamiR for EDN2 induction. Intriguingly, VEGFA transcripts were reduced by both miR-210-mimic and -inhibitor, demonstrating that EDN2 and VEGFA are distinctly regulated. Disrupted hypoxic response in the GLCs of periovulatory follicles in PCOS women may play a role in ovulation failure, and in the reduced fertility prevalent in this syndrome.

scholarly journals Intercellular calcium signaling via gap junction in connexin-43-transfected cells.

1998 ◽  
Vol 273 (35) ◽  
pp. 22856
Author(s):  
Toshihiko Toyofuku ◽  
Masanori Yabuki ◽  
Kinya Otsu ◽  
Tsunehiko Kuzuya ◽  
Masatsugu Hori ◽  
...  
Keyword(s):  
Calcium Signaling ◽  
Gap Junction ◽  
Connexin 43 ◽  
Transfected Cells

scholarly journals Desensitization of neuromedin B receptors (NMB-R) on native and NMB-R-transfected cells involves down-regulation and internalization.

1994 ◽  
Vol 269 (16) ◽  
pp. 11721-11728
Author(s):  
R.V. Benya ◽  
T. Kusui ◽  
F. Shikado ◽  
J.F. Battey ◽  
R.T. Jensen
Keyword(s):  
Down Regulation ◽  
Transfected Cells ◽  
Neuromedin B

scholarly journals miR-21-5p regulates mitochondrial respiration and lipid content in H9C2 cells

2019 ◽  
Vol 316 (3) ◽  
pp. H710-H721 ◽  
Author(s):  
Victoria L. Nasci ◽  
Sandra Chuppa ◽  
Lindsey Griswold ◽  
Kathryn A. Goodreau ◽  
Ranjan K. Dash ◽  
...  
Keyword(s):  
Lipid Peroxidation ◽  
Fatty Acid ◽  
Palmitic Acid ◽  
Fatty Acid Oxidation ◽  
Lipid Content ◽  
Mitochondrial Respiration ◽  
Acid Oxidation ◽  
H9c2 Cells ◽  
Cellular Lipid ◽  
Transfected Cells

Cardiovascular-related pathologies are the single leading cause of death in patients with chronic kidney disease (CKD). Previously, we found that a 5/6th nephrectomy model of CKD leads to an upregulation of miR-21-5p in the left ventricle, targeting peroxisome proliferator-activated receptor-α and altering the expression of numerous transcripts involved with fatty acid oxidation and glycolysis. In the present study, we evaluated the potential for knockdown or overexpression of miR-21-5p to regulate lipid content, lipid peroxidation, and mitochondrial respiration in H9C2 cells. Cells were transfected with anti-miR-21-5p (40 nM), pre-miR-21-5p (20 nM), or the appropriate scrambled oligonucleotide controls before lipid treatment in culture or as part of the Agilent Seahorse XF fatty acid oxidation assay. Overexpression of miR-21-5p attenuated the lipid-induced increase in cellular lipid content, whereas suppression of miR-21-5p augmented it. The abundance of malondialdehyde, a product of lipid peroxidation, was significantly increased with lipid treatment in control cells but attenuated in pre-miR-21-5p-transfected cells. This suggests that miR-21-5p reduces oxidative stress. The cellular oxygen consumption rate (OCR) was increased in both pre-miR-21-5p- and anti-miR-21-5p-transfected cells. Levels of intracellular ATP were significantly higher in anti-mR-21-5p-transfected cells. Pre-miR-21-5p blocked additional increases in OCR in response to etomoxir and palmitic acid. Conversely, anti-miR-21-5p-transfected cells exhibited reduced OCR with both etomoxir and palmitic acid, and the glycolytic capacity was concomitantly reduced. Together, these results indicate that overexpression of miR-21-5p attenuates both lipid content and lipid peroxidation in H9C2 cells. This likely occurs by reducing cellular lipid uptake and utilization, shifting cellular metabolism toward reliance on the glycolytic pathway. NEW & NOTEWORTHY Both overexpression and suppression of miR-21-5p augment basal and maximal mitochondrial respiration. Our data suggest that reliance on glycolytic and fatty acid oxidation pathways can be modulated by the abundance of miR-21-5p within the cell. miR-21-5p regulation of mitochondrial respiration can be modulated by extracellular lipids.

scholarly journals SSADH Variants Increase Susceptibility of U87 Cells to Mitochondrial Pro-Oxidant Insult

2020 ◽  
Vol 21 (12) ◽  
pp. 4374
Author(s):  
Giovanna Menduti ◽  
Alessandra Vitaliti ◽  
Concetta Rosa Capo ◽  
Daniele Lettieri-Barbato ◽  
Katia Aquilano ◽  
...  
Keyword(s):  
Lipid Peroxidation ◽  
Mitochondrial Function ◽  
Antioxidant Defense ◽  
Mitochondrial Morphology ◽  
Common Snps ◽  
Protein Markers ◽  
Triple Mutant ◽  
Transfected Cells ◽  
Semialdehyde Dehydrogenase ◽  
Succinate Semialdehyde

Succinate semialdehyde dehydrogenase (SSADH) is a mitochondrial enzyme, encoded by ALDH5A1, mainly involved in γ-aminobutyric acid (GABA) catabolism and energy supply of neuronal cells, possibly contributing to antioxidant defense. This study aimed to further investigate the antioxidant role of SSADH, and to verify if common SNPs of ALDH5A1 may affect SSADH activity, stability, and mitochondrial function. In this study, we used U87 glioblastoma cells as they represent a glial cell line. These cells were transiently transfected with a cDNA construct simultaneously harboring three SNPs encoding for a triple mutant (TM) SSADH protein (p.G36R/p.H180Y/p.P182L) or with wild type (WT) cDNA. SSADH activity and protein level were measured. Cell viability, lipid peroxidation, mitochondrial morphology, membrane potential (ΔΨ), and protein markers of mitochondrial stress were evaluated upon Paraquat treatment, in TM and WT transfected cells. TM transfected cells show lower SSADH protein content and activity, fragmented mitochondria, higher levels of peroxidized lipids, and altered ΔΨ than WT transfected cells. Upon Paraquat treatment, TM cells show higher cell death, lipid peroxidation, 4-HNE protein adducts, and lower ΔΨ, than WT transfected cells. These results reinforce the hypothesis that SSADH contributes to cellular antioxidant defense; furthermore, common SNPs may produce unstable, less active SSADH, which could per se negatively affect mitochondrial function and, under oxidative stress conditions, fail to protect mitochondria.

scholarly journals Functional, Structural, and Distribution Analysis of the Chorionic Gonadotropin Receptor Using Murine Monoclonal Antibodies

2003 ◽  
Vol 88 (11) ◽  
pp. 5537-5546 ◽  
Author(s):  
Ada Funaro ◽  
Anna Sapino ◽  
Bruna Ferranti ◽  
Alberto L. Horenstein ◽  
Isabella Castellano ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Tumor Cell ◽  
Cell Lines ◽  
Chorionic Gonadotropin ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster ◽  
Signal Pathways ◽  
Distribution Analysis ◽  
Tumor Cell Lines ◽  
Transfected Cells

Abstract LH and human chorionic gonadotropin (hCG) control steroid production and gametogenesis. They also function as growth factors through interaction with a specific receptor that is a member of the seven-transmembrane receptor family coupled via G proteins to signal pathways involving cAMP and phospholipase C/inositol 3 phosphate. For this study, monoclonal antibodies (mAbs) were raised against the human LH receptor (LHR)/hCG receptor (hCGR), using Chinese hamster ovary LHR-transfected cells as the immunogen. Two reagents were then selected on the basis of their ability to recognize the full-length transmembrane re-ceptor expressed both by Chinese hamster ovary LHR-transfected cells and by a limited number of tumor cell lines. One of these mAbs reacts with the LHR/hCGR in tissue sections of both frozen and paraffin-embedded specimens. This unique feature allowed us to map the cytological distribution of LHR/hCGR in human breast tissues at different stages of development in physiological and benign pathological conditions. The same mAb proved to be agonistic: receptor ligation elicits signals that modulate the growth of selected breast tumor cell lines. This observation suggests that the mAb recognizes an epitope that is included in the domain of the receptor involved in the interaction with the natural ligand.

Sign in / Sign up

Export Citation Format

Share Document