Volume 10, Issue 4 (Vol.10 No.4 Jan 2022)
rbmb.net 2022, 10(4): 518-526 |
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Department of Pathological Analysis, College of Science, University of Thi-Qar, Thi-Qar, 64001 Iraq.
Abstract: (2741 Views)
Background: Phosphatidylinositol 3,4,5-trisphosphate [PtdIns (3,4,5) P3) and Phosphatidylinositol 4,5-trisphosphate (PtdIns (4,5) P2] form an insignificant number of phospholipids but play important roles in controlling membrane-bound signalling. Little attention has been given to visualize and monitor changes or differences in the local generation of PtdIns (4,5) P2 and PtdIns (3,4,5) P3 in the cell membranes of MDAMB- 231 breast cancer cell lines.
Methods: PLCδ1-PH-GFP and Btk-PH-GFP were used as biosensors to detected PtdIns (4,5) P2 and PtdIns(3,4,5)P3 respectively. These biosensors and antibodies were transfected, immuostained and then visualized by confocal microscopy on different cell surfaces.
Results: Our results showed that PLCδ1-PH-GFP/mCherry was localized at the cell membrane, while Btk-PH-GFP/mCherry was sometimes localized at the cell membrane but there was also a large amount of fluorescence present in the cytosol and nucleus. Our results also showed that the cells that expressed low levels of Btk-PH-GFP the fluorescence was predominantly localised to the cell membrane. While the cells that expressed high levels of Btk-PH-GFP the fluorescence was localization in the cytosol and cell membrane. Our results demonstrated that both anti-PtdIns(4,5)P2 and anti-PtdIns(3,4,5)P3 antibodies were localized everywhere in cell.
Conclusions: Our results suggest that PLCδ1-PH-GFP and Btk-PH-GFP/mCherry have more specificity, reliability, suitability and accuracy than antibodies in binding with and detecting PtdIns(4,5)P2 and PtdIns (3,4,5)P3 and in studying the molecular dynamics of phospholipids in live and fixed cells.
Methods: PLCδ1-PH-GFP and Btk-PH-GFP were used as biosensors to detected PtdIns (4,5) P2 and PtdIns(3,4,5)P3 respectively. These biosensors and antibodies were transfected, immuostained and then visualized by confocal microscopy on different cell surfaces.
Results: Our results showed that PLCδ1-PH-GFP/mCherry was localized at the cell membrane, while Btk-PH-GFP/mCherry was sometimes localized at the cell membrane but there was also a large amount of fluorescence present in the cytosol and nucleus. Our results also showed that the cells that expressed low levels of Btk-PH-GFP the fluorescence was predominantly localised to the cell membrane. While the cells that expressed high levels of Btk-PH-GFP the fluorescence was localization in the cytosol and cell membrane. Our results demonstrated that both anti-PtdIns(4,5)P2 and anti-PtdIns(3,4,5)P3 antibodies were localized everywhere in cell.
Conclusions: Our results suggest that PLCδ1-PH-GFP and Btk-PH-GFP/mCherry have more specificity, reliability, suitability and accuracy than antibodies in binding with and detecting PtdIns(4,5)P2 and PtdIns (3,4,5)P3 and in studying the molecular dynamics of phospholipids in live and fixed cells.
Type of Article: Original Article |
Subject:
Cell Biology
Received: 2021/09/5 | Accepted: 2021/09/19 | Published: 2022/02/7
Received: 2021/09/5 | Accepted: 2021/09/19 | Published: 2022/02/7
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