PP2A promotes dephosphorylation of Akt that associates with eNOS. BAECs were incubated ± pal ± OA. For the last 10 min, cells
were treated with veh or ins (A–D). After eNOS immunoprecipitation (IP), Western blot (IB) for PP2A, Akt, p-Akt, and eNOS was performed. A: PP2A association with eNOS (n = 12–34 per treatment). B: Akt association with eNOS (n = 13–35 per treatment). C: p-Akt Ser473 and p-Akt Thr308 association with eNOS (n = 6–8 per treatment). D: p-eNOS Ser1177 to total eNOS (n = 12–18 per treatment). *P < 0.05 vs. veh, #P < 0.05 vs. (−) pal (+) ins (−) OA. E: BAECs were incubated ± pal ± myr. After PP2A IP, IB for I2PP2A and PP2A was performed. I2PP2A that coimmunoprecipitated
with PP2A was decreased by pal in a ceramide-dependent manner (n = 15–17 per treatment). A reciprocal IP is shown in Supplementary Fig. 9C. *P < 0.05 vs. (−) myr (−) pal; #P < 0.05 vs. (−) myr (+) pal. Results represent mean ± SEM. Pal, palmitate; myr, myriocin; veh, vehicle; ins, insulin.