Brefeldin A problem
Ian A. York
york at mbcrr.dfci.harvard.edu
Fri Jan 27 21:28:25 EST 1995
I'd appreciate suggestions from people who have worked with
brefeldin A. We want to block glycoprotein transport in a T cell line.
To test the function of the BFA, we activated the cells, in the presence
of BFA, and measured the surface appearance of activation markers. The
one we have looked at most is produced de novo on activation, so if the
BFA is working there should be no apparent upregulation.
To our surprise, there was a significant population that showed
levels as bright as in the absence of BFA. We then tried increasing
levels of BFA (from 5 to 50 ug/ml) (5 ug/ml is our usual concentration).
We were even more surprised.
At the lowest concentration, there was a clear biphasic
population - that is, either expression was blocked altogether (about
half the cells), or it was expressed normally (the other half). For a
couple of higher concentration points, the proportion of negatives
increased, to a maximum of about 70%; again, the remainder showed normal
intensity. Then, as the BFA concentration increased more, the cells
shift back to normal intensity. At 20 ug/ml, over 75% of the cells are
of normal intensity. At 50 ug/ml, the population is no longer biphasic.
We also tried pretreating the cells, though only at a low
concentration of BFA; it made no difference compared to treating
simulataneously with the activation.
I'm puzzled. Anyone got any suggestions?
Ian York (york at mbcrr.harvard.edu)
Dana-Farber Cancer Institute, 44 Binney St., Boston MA 02115
Phone (617)-632-4328 Fax (617)-632-2627
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