Figure 3.
Figure 3. Dab2 recruitment to apoER2 via the NPXY motif is required for aPL induction of PP2A activity by LCMT-1 interaction with PP2A-C in endothelial cells, and for resulting eNOS antagonism. (A) HAECs were treated with vehicle, NHIgG, or aPLs (100 µg/mL) for 90 minutes, cell lysates were prepared, apoER2 was immunoprecipitated, and the co-IP of β2GPI and Dab2 was evaluated by immunoblotting. (B-C) HAECs were transfected with control RNAi (Con RNAi) or RNAi targeting apoER2 or Dab2; 24 hours later they were treated with BBG or 3F8 (10 µg/mL), or NHIgG or aPL (100 µg/mL) for 90 minutes, and lysates were prepared to detect apoER2 and Dab2 by immunoblotting (B) or to quantify PP2A activity (C, n = 4). (D-E) HAECs were transfected with control RNAi or RNAi targeting Dab2; 24 hours later, they were treated with NHIgG or aPLs, and vehicle or VEGF (100 ng/mL, 30 minutes), and immunoblotting was done to detect eNOS S1177 phosphorylation (peNOS), total eNOS, and GAPDH (D). With the same treatments, NOS activity was determined in intact cells (E, n = 6-12). (F-I) Endogenous apoER2 was silenced with RNAi, and using adenoviral constructs HA-tagged WT apoER2 (WT) or mutant forms of apoER2 harboring NFDNPVA (NPVA) or EIGNPVY (EIG) substitutions for NFDNPVA were reintroduced (F). Cells were treated 24 hours later with NHIgG or aPLs, and vehicle or VEGF; apoER2 was immunoprecipitated using anti-HA antibody, and Dab2 co-IP was evaluated by immunoblotting (G). In parallel studies, PP2A activity was quantified in cell lysates (H, n = 4), NOS activity was evaluated in intact cells (I, n = 8-16), and PP2A-C subunit methylation at L309 was assessed in cell lysates by immunoblotting (I). (J) Following control RNAi or RNAi silencing Dab2, 24 hours later, cells were treated with NHIgG or aPLs, and vehicle or VEGF; (K) PP2A-C was immunoprecipitated; and PP2A-C metL309 or total PP2A-C were detected by immunoblotting. The co-IP of LCMT-1 was also evaluated by immunoblotting. Values in graphs are mean ± SEM, ****P < .0001, and findings in all immunoblots were confirmed in 3 independent experiments. (L) Summary of the findings in the figure.

Dab2 recruitment to apoER2 via the NPXY motif is required for aPL induction of PP2A activity by LCMT-1 interaction with PP2A-C in endothelial cells, and for resulting eNOS antagonism. (A) HAECs were treated with vehicle, NHIgG, or aPLs (100 µg/mL) for 90 minutes, cell lysates were prepared, apoER2 was immunoprecipitated, and the co-IP of β2GPI and Dab2 was evaluated by immunoblotting. (B-C) HAECs were transfected with control RNAi (Con RNAi) or RNAi targeting apoER2 or Dab2; 24 hours later they were treated with BBG or 3F8 (10 µg/mL), or NHIgG or aPL (100 µg/mL) for 90 minutes, and lysates were prepared to detect apoER2 and Dab2 by immunoblotting (B) or to quantify PP2A activity (C, n = 4). (D-E) HAECs were transfected with control RNAi or RNAi targeting Dab2; 24 hours later, they were treated with NHIgG or aPLs, and vehicle or VEGF (100 ng/mL, 30 minutes), and immunoblotting was done to detect eNOS S1177 phosphorylation (peNOS), total eNOS, and GAPDH (D). With the same treatments, NOS activity was determined in intact cells (E, n = 6-12). (F-I) Endogenous apoER2 was silenced with RNAi, and using adenoviral constructs HA-tagged WT apoER2 (WT) or mutant forms of apoER2 harboring NFDNPVA (NPVA) or EIGNPVY (EIG) substitutions for NFDNPVA were reintroduced (F). Cells were treated 24 hours later with NHIgG or aPLs, and vehicle or VEGF; apoER2 was immunoprecipitated using anti-HA antibody, and Dab2 co-IP was evaluated by immunoblotting (G). In parallel studies, PP2A activity was quantified in cell lysates (H, n = 4), NOS activity was evaluated in intact cells (I, n = 8-16), and PP2A-C subunit methylation at L309 was assessed in cell lysates by immunoblotting (I). (J) Following control RNAi or RNAi silencing Dab2, 24 hours later, cells were treated with NHIgG or aPLs, and vehicle or VEGF; (K) PP2A-C was immunoprecipitated; and PP2A-C metL309 or total PP2A-C were detected by immunoblotting. The co-IP of LCMT-1 was also evaluated by immunoblotting. Values in graphs are mean ± SEM, ****P < .0001, and findings in all immunoblots were confirmed in 3 independent experiments. (L) Summary of the findings in the figure.

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