PECAM-1 signaling is involved in SK-mediated cell survival. The effect of altering PECAM-1 signaling on cell survival as measured by the MTS assay of ECs^SK (▪) and ECs^EV (□) in (A) suspension and in (B) serum-free conditions. The effect on cell survival of 20 μg/mL rabbit polyclonal anti–PECAM-1 antibody (RP) and 20 μg/mL of 6F6 added at plating. Panel A shows the pooled data of 10 observations from 2 separate experiments normalized to day 0 = 1. Bars represent 95% confidence intervals. *P less than .001 compared with normal rabbit serum (NRS)–treated corresponding ECs^EV or ECs^SK at day 2. Panel B shows the results of 1 experiment representative of 3 performed. □ indicates no antibody; ▪, polyclonal antibody; and ▦, monoclonal 6F6. Mean ± SEM of quadruplicate determinations is given. *P less than .001 compared with ECs^SK at day 2. (C) Western blot showing phosphorylation of PECAM (P-PECAM) in normal ECs after treatment for 10 minutes with control (Nil), the monoclonal antibody (6F6), or polyclonal anti-PECAM antibody (RP). Total PECAM is given as a loading control (bottom). Cells were grown in normal medium. (D) Western blot of the phosphorylation of PECAM-1 (P-PECAM) in ECs^EV and ECs^SK after growth for 6 hours in SF conditions and then 10 minutes treatment with either control or anti-PECAM antibodies. Actin is given as a loading control. (E) The effect of incubation of antibodies to PECAM on the cell surface expression of PECAM on normal ECs, ECs^EV, and ECs^SK. Cells were incubated under SF conditions with either no treatment, NIL (□), polyclonal rabbit antibody to PECAM (RP; ▪), or monoclonal antibody to PECAM (6F6; ▨) for 6 hours, harvested, and stained for flow cytometry analysis. The data for the mean ± SEM pooled from 4 experiments. *P less than .001 compared with no antibody control. Inset shows a Western blot for PECAM in ECs^SK after antibody treatment and probed with a goat anti-PECAM antibody. Similar effects were seen in ECs^EV (not shown).