Supplementary Figure 3: PP2A regulates PDE4D5 phosphorylation and inflammation.

(a) Identification of PP2A as a PDE4D5 binding protein. FLAG-PDE4D5 expressing BAECs on FN were lysed and immunoprecipitated with FLAG antibody. Bound protein with size of 35 kDa was submitted for mass analysis and identified as PP2A catalytic subunit. (b) BAECs expressing FLAG-tagged PDE4D5 were plated on either FN or matrigel for indicated times. PDE4D5 was immunoprecipitated with FLAG antibody and eluted with FLAG peptides. Similar results were obtained in 3 experiments. (c) BAECs expressing PDE4D5 wild type were kept in suspension for 90 min then replated on FN-coated dishes for the indicated times. For okadaic acid treatment, cells in suspension were added with 5 nM OA for last 20 min before replating on FN. S651 phosphorylation was assayed by Western blotting (n = 4 independent experiments). (d) BAECs expressing PDE4D5 were plated on FN for 5 hr then pretreated with DMSO or okadaic acid (OA, 5 nM) and then stimulated with IL1β for 30 min. S651 phosphorylation was assayed by Western blotting (n = 3 independent experiments). (e) BAECs expressing PDE4D5 were transfected with a siRNAs targeting PP2A catalytic subunit. The cells were replated on FN then subject to laminar shear for 30 min. (f) BAECs were plated on FN for 5 hr then pretreated with DMSO or okadaic acid (OA, 5 nM) and then stimulated with IL1β for 30 min. NFκB activity was assayed by Western blotting (n = 3 independent experiments). (g) BAECs were transfected with two different siRNAs targeting the PP2A catalytic subunit. The cells were replated on FN then subject to oscillatory shear for 2 hrs (n = 3 independent experiments). Data are represented as means ± SEM. ^∗p < 0.05 by one way ANOVA (d,g) or two-tailed t-test (c,f). Source data are provided in Supplementary Table 1.