P66 SHC1 recruitment to apoER2 via the proline-rich C terminus of the receptor is required for PP2A-A recruitment in response to aPL, and the resulting activation of PP2A and eNOS antagonism in endothelial cells. (A) HAECs were treated with NHIgG or aPLs (100 µg/mL) for 90 minutes, and with vehicle or VEGF (100 ng/mL) for 30 minutes, apoER2 was immunoprecipitated, and the co-IP of Dab2, PP2A-C, PP2A-A, and p66 SHC1 was evaluated by immunoblotting. (B) HAECs were transfected with control RNAi or RNAi targeting Dab2; 24 hours later, they were treated with NHIgG or aPLs, and vehicle or VEGF, apoER2 was immunoprecipitated, and the co-IP of PP2A-A and p66 SHC1 was evaluated by immunoblotting. (C) Using a parallel approach, SHC1 was silenced; 24 hours later, cell treatments occurred and apoER2 was immunoprecipitated, and the co-IP of Dab2 and PP2A-A was evaluated. (D-F) Following SHC1 silencing and NHIgG vs aPLs and vehicle vs VEGF treatment, PP2A activity (D, n = 6-12) or eNOS S1177 phosphorylation (E) was evaluated in cell lysates, or NOS activity was assessed in intact cells (F, n = 6-12). (G) Endogenous apoER2 was silenced with RNAi, and using adenoviral constructs HA-tagged WT apoER2 (WT) or mutant forms of apoER2 harboring NFDNPVA (NPVA) substitution for NFDNPVA or lacking the proline-rich C terminus of the receptor (Δ59) were reintroduced. Cells were treated 24 hours later with NHIgG or aPLs, and vehicle or VEGF, apoER2 was immunoprecipitated using anti-HA antibody, and SHC1 co-IP was evaluated by immunoblotting. (H-I) In parallel studies, PP2A activity was quantified in cell lysates (H, n = 4) or NOS activity was evaluated in intact cells (I, n = 8-16). Values in graphs are mean ± SEM, ****P < .0001, and findings in all immunoblots were confirmed in 3 independent experiments. (J) Summary of the findings in the figure.