APL promote PP2A-C L309 methylation and Y307 dephosphorylation, PP2A subunit assembly, and PP2A-B Bδ and B′α recruitment to Akt and eNOS as selective phosphoprotein substrates in endothelial cells. (A) HAECs were treated with NHIgG, aPLs (100 µg/mL), BBG or 3F8 (10 µg/mL) in the presence of the inactive analog Dm-endo or endothall (10 µM) for 90 minutes, and with vehicle (PBS) or VEGF (100 ng/mL) for 30 minutes, cell lysates were prepared, and immunoblot analysis was performed detecting methylated PP2A-C L309 (metL309), phosphorylated PP2A-C Y307 (pY307), total PP2A-C and GAPDH. (B) HAECs were treated with NHIgG or aPLs (100 µg/mL) for 90 minutes, and with vehicle (PBS) or VEGF (100 ng/mL) for 30 minutes, and immunoblot analysis was performed detecting methylated PP2A-C L309 (metL309), phosphorylated PP2A-C Y307 (pY307), total PP2A-C, and GAPDH. Immunoblotting was also performed for Akt phosphorylation at S473 (p-Akt), total Akt (Akt), phosphorylated eNOS at S1177 (peNOS), and total eNOS (eNOS). (C) In parallel studies, PP2A-C was immunoprecipitated, and the co-IP of PP2A-A and LCMT-1 was evaluated along with PP2A-C metL309. (D) Reverse transcription–polymerase chain reaction was performed to detect transcripts for PP2A-B subunits Bα, Bβ, Bγ, Bδ, B′α, B′β, and B′ε in HAECs. (E) HAECs were transfected with control RNAi or RNAi targeting PP2A-Aα, or PP2A-B subunit Bα, Bδ, B′α, B′β, or B′ε; 24 hours later, the cells were treated with aPLs or NHIgG (100 µg/mL) for 90 minutes, and PP2A activity was quantified in cell lysates; N = 4. Values are mean ± SEM, ****P < .0001. (F-H) HAECs were treated as in panel B, Akt or eNOS was immunoprecipitated, and the co-IP of PP2A-A, PP2A-C metL309, Bδ, B′α, and B′β was evaluated along with immunoblotting for pAkt and peNOS. Findings in all immunoblots were confirmed in 3 independent experiments. (I) Summary of the findings in the figure.