Leakage of proteins from digitonin-permeabilized platelets.

Platelets were incubated with 15 μM of digitonin in K⁺-glutamate buffer for different periods of times. Platelets were then recovered by centrifugation, and pellets (P) were resuspended in K⁺-glutamate buffer to the same volumes of the corresponding supernatants (S; incubation media). Equal aliquots (1:1) of P and S were run on SDS-PAGE, followed by immunoblotting with antibodies against PKC, pleckstrin, and MARCKS. Under these conditions, MARCKS was not detected in the medium (S). Therefore, S was concentrated 30 times; aliquots were run again and tested for the presence of MARCKS (1:30). In this case, a strong band was observed, and this is shown at the bottom of the figure. The degree of platelet permeabilization in this experiment was determined by incubating fixed platelets for 15 minutes with rhodamine-phalloidin. Results obtained for intact (Int.) and permeabilized (Per.) platelets after 5 and 20 minutes of digitonin treatment are shown at the top of the figure. Phase contrast (PC) and fluoresence (F) fields of the preparations are shown at left and right, respectively.