Figure 11.

PI3K activity correlates with constitutive macropinocytosis. (A) PI3K was assayed in situ and compared with peroxidase accumulation. The indicated cells were incubated for 6 h in phosphate-free DMEM containing 0.5% dialyzed FCS and 250 μCi/ml [³²P]orthophosphate. Inhibitors were added 30 min before this incubation and maintained throughout. After a brief wash, cell lipids were extracted and phosphoinositides were analyzed by HPLC and expressed in absolute counts. ND, not detectable. Data for PtdIns(3,4)P2 levels are from one experiment representative of two to five experiments. Counts of PtdIns(3,4,5)P3 are from the same experiment, which provided the best resolution of this minor compound. These values are compared with macropinocytosis, measured after cells were incubated for 30 min in DMEM without serum in the presence or absence of the inhibitors and then for another 30 min with HRP, as in Figure 1. Values are means ± SEM of 16 to 89 dishes compiled from all experiments performed for this paper. (B) PI3K was assayed in vitro in the indicated cell lines after IRS-1 immunoprecipitation. Values are means ± SEM of five to nine dishes per condition.