Fig 2: Effects of purified Epac1 protein on UBC9 SUMO thioester formation.In vitro SUMO thioester intermediate formation of recombinant UBC9 in the presence or absence of Epac1 or Epac1 plus cAMP.

Fig 3: Epac and PKA synergise to inhibit VSMC cell-cycle progression. Asynchronous VSMC were stimulated with 200 µM BNZ, 200 µM CPT, 200 µM BNZ plus 200 µM CPT, or 200 µM db-cAMP: A, for 24 h with the last 6 h in the presence of 10 µM BrdU and analysed for BrdU incorporation, n = 6; B and C, for 18 h and cell lysates analysed by western blotting as indicated, n = 3. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001 versus control.

Fig 4: ox-LDL and 007-AM promote cellular SUMOylation and UBA2 activation in an Epac1-dependent manner.Cellular SUMOylation probed by anti-SUMO2/3 antibody in WT and Epac1-/- (KO) BMDMs treated with vehicle or ox-LDL (40 µg/ml) for 7 min (A) or with vehicle or 5 µM 007-AM for 30 min (B). Formation of UBA2-SUMO thioester (UBA2*) in WT and Epac1-/- BMDMs in response to ox-LDL (C) or 007-AM stimulation (D). (E) Representative images and quantification of ORO-stained mouse primary macrophages treated with ox-LDL (40 µg/ml) in the presence or absence of the UBA2 inhibitor ML-792. Data are shown as means ± SEM.

Fig 5: Selective activation of PKA and Epac signalling with N6-Benzoyl-cAMP and 8-CPT-2'-O-Me-cAMP, respectively. VSMC were serum deprived for 24 h and stimulated with 200 µM BNZ, CPT, BNZ plus CPT or 100 µM forskolin for 30 min as indicated. A, Cell lysates were analysed for PKA activity by kemptide phosphorylation assay. Upper bands represent PKA phosphorylated kemptide while lower bands are un-phosphorylated kemptide. Recombinant PKA catalytic subunit and lysis buffer were included in the assay as positive and negative controls respectively. B, Cell lysates were analysed for phospho-VASP (slower migrating band) by western blotting with a total VASP antibody, n = 3. C, Rap1 activity was quantified by Ral-GDS pulldown, n = 6. * indicates p < 0.05 versus control.