Biochemical and Biophysical Research Communications
Localization of P-glycoprotein at the nuclear envelope of rat brain cells
Section snippets
Antibodies
The murine monoclonal P-gp antibodies C219 and MRK16 were purchased from ID Laboratories (London, ON) and Kamiya Biomedical (Seattle, WA), respectively. The C219 antibody recognizes a conserved intracellular epitope (VQEALD) on all human and rodent isoforms of P-gp [20] while the MRK-16 antibody recognizes a conserved extracellular epitope on all drug-transporting isoforms of human and rat P-gp protein [20]. Protein A–gold was prepared with 10 mm gold particle according to our previously
Nuclear localization of P-gp by immunofluorescence
Immunofluorescence using P-gp monoclonal C219 antibody revealed a signal at the plasma membranes of RBE4 and MLS-9 cells respectively (Fig. 1). Signal was also detected subcellularly in the cytoplasm, as well as at the nuclear envelope of both cell types. No signal was detected in the control samples in which incubation with the primary antibody was substituted with PBS (data not shown).
Morphology of isolated nuclei
To determine the nuclear localization of P-gp in RBE4 and MLS-9 cells, nuclei were first isolated and the
Discussion
Drug efflux transport proteins are primarily expressed at the plasma membrane of endothelial cells and exert their action by pumping substrate drugs out of the cell [4]. The role of P-gp mediated cellular drug resistance in brain pathologies is compelling because this transport protein is present at the BBB and restricts access of drugs to the brain parenchyma. However, brain parenchymal cells including neurons and glial (microglia, astrocytes) cells can also express P-gp [27]. Thus, once a
Acknowledgments
This work is supported by a grant from the Canadian Institutes of Health Research (MOP 56976) and the Ontario HIV Treatment Network, Ontario Ministry of Health. The authors would like to thank Ms. Manisha Ramaswamy and Ms. Diane Gingras for their excellent technical assistance and Dr. Jeff Henderson for the help and advice with the undertaking of the fluorescence microscopy studies.
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