Time course of phosphorylation of PECAM-1 in HUVEC in response to incubation with SS RBC. HUVEC were labeled with³²P and incubated with SS RBC (2% Hct) for the indicated time period and then processed for immunoprecipitation with PECAM-1 antibody. The immunoprecipitate was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and radioactivity quantitated in the gel lane corresponding to PECAM-1 (130 kD) by Ambis radiogel scanner as described in Materials and Methods. The data is presented as percent increase in the incorporation of ³²P in PECAM-1, assigning the value of 100% for ³²P incorporated into PECAM-1 in untreated HUVEC. Data are mean ± SD of n = 3, with each experiment run in duplicate for indicated time points, except for 1-hour period (n = 7). ³²P incorporated into PECAM-1 was 638% ± 220% at 1-hour time point, with a range of 410% to 850%. The replicate data for the same donor sample showed less than 15% difference in the ³²P incorporation into PECAM-1.