basal sheets of mdck cells transiently expressing mgfp - h - rasg12v were prepared exactly as described . the prepared basal pm sheets on gold em grids were washed and fixed , and the cytosolic leaflet was labeled with anti - gfp antibody conjugated to 4.5-nm gold particles as previously described . digital images of the immunogold - labeled plasma membrane sheets were taken in a jeol 1400 transmission em . intact 1 m areas of the plasma membrane sheet were identified using imagej , and the x , y coordinates of the gold particles were determined . k - functions were calculated and standardized on the 99% confidence interval for complete spatial randomness . bootstrap tests to examine differences between replicated point patterns were constructed exactly as described previously , and statistical significance evaluated against 1,000 bootstrap samples . basal sheets of mdck cells transiently expressing mgfp - h - rasg12v were prepared exactly as described . the prepared basal pm sheets on gold em grids were washed and fixed , and the cytosolic leaflet was labeled with anti - gfp antibody conjugated to 4.5-nm gold particles as previously described . digital images of the immunogold - labeled plasma membrane sheets were taken in a jeol 1400 transmission em . intact 1 m areas of the plasma membrane sheet were identified using imagej , and the x , y coordinates of the gold particles were determined . k - functions were calculated and standardized on the 99% confidence interval for complete spatial randomness . bootstrap tests to examine differences between replicated point patterns were constructed exactly as described previously , and statistical significance evaluated against 1,000 bootstrap samples .
the ras gtpases comprising three main isoforms h- , n- and k - ras operate at the plasma membrane as molecular switches in essential signaling pathways . active concentration of the minor phospholipid phosphatidylserine in the inner leaflet of the plasma membrane contributes to the electrostatic potential that is required for k - ras anchoring to the plasma membrane . 
 we recently observed that staurosporine and related analogs : 7-oxostaurosporine , ucn-01 and ucn-02 , long known as relatively non - specific protein kinase inhibitors , block endosomal sorting and recycling of phosphatidylserine , resulting in redistribution of phosphatidylserine to endosomes and endomembranes with concomitant mislocalization of k - ras . 
 staurosporines are therefore a new tool to study phosphatidylserine trafficking . 
 we discuss whether the mechanism of action of ucn-01 , an fda - approved staurosporine analog used as an anti - cancer therapeutic , is related to effects on phosphatidylserine subcellular distribution . given the high prevalence of expression of constitutively active k - ras in human cancers , we ask whether inhibitors of phosphatidylserine trafficking may have important therapeutic applications .