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To measure translocase activity, fluorescent NBD-PS or NBD-PC derivatives are introduced into the outer leaflet of cells, and the kinetics of probe transfer to the inner leaflet measured periodically by first removing probe still on the surface with BSA-containing buffers and then quantitating the remaining, internalized probe by measuring cellular fluorescence using flow cytometry.
Calreticulin is exposed on the outer leaflet of cells during the early phase of cell death following treatment with anthracyclins.
This is precisely the range over which PIP2 is known to vary in the inner membrane leaflet of cells (Balla, 2013).
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Indeed, PS is mainly found in the inner leaflet of cell membrane.
The time-dependent release of [H]-Etn metabolites to the medium by all unstimulated cell lines reflects the normal basal turnover of EtnPGs concentrated in the cytosolic leaflet of cell membranes (e.g., Vance 2008).
Because PA is a naturally occurring 'signaling lipid' in the inner leaflet of cell membranes, we think we have likely uncovered a new and biologically relevant mode of Kv channel regulation.
Thus, we conclude that, as might be anticipated from the restriction of PIP2 to the inner leaflet of cell membranes, PIP2 primarily interacts with the intracellular surface of Kir channels.
Moreover, the steepest portion of the graph (corresponding to a few tenths to one percent PIP2) is in agreement the known concentration range of PIP2 in the inner leaflet of cell membranes (Heck et al., 2007; van Meer et al., 2008; Balla, 2013).
This further demonstrates that PS exposure to the outer lipid leaflet of cancer cells is not caused by cell culturing procedures but is an original and permanent characteristic of cancer cells.
Experimental data show that zwitterionic lipid headgroups take part in the interaction with Penetratin suggesting that the external leaflet lipids of cells plasma membrane are competent for peptide interaction in the absence of net negative charges.
Furthermore, we proved that not only do cell lines derived from primary lesions and metastasis show PS on the outer leaflet of the cell membrane but also cells from primary cancer cell cultures.
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Justyna Jupowicz-Kozak
CEO of Professional Science Editing for Scientists @ prosciediting.com