Skip to main content
. 2019 Feb;1866(2):264–276. doi: 10.1016/j.bbamcr.2018.11.004

Fig. 2.

Fig. 2

I942 activates EPAC1-dependent signalling in HUVECs.

a) HUVECs were stimulated for 5 h with the indicated combinations of 10 μM forskolin, 10 μM rolipram, 100 nM ADM2 and 100 μM I942 in the presence 10 μM MG132. Cell extracts were then prepared and immunoblotted with anti-SOCS3 and anti-GAPDH antibodies. The histogram in the lower panel demonstrates changes in SOCS3 expression relative to control cells for three separate experiments. Significant increases in SOCS3 protein expression in I942-treated cells are indicated; ***, p < 0.001 (n = 3). Non-significant changes in SOCS3 immunoreactivity in cells treated with I942 and forskolin are also indicated (ns).

b) Confluent HUVECs were pre-incubated with siRNA to EPAC1 or non-targeting, scrambled siRNA for 24 h, after which cells were treated with the proteasome inhibitor, 10 μM MG132 (to prevent breakdown of cellular SOCS3 protein), and then stimulated for 5 h in the presence or absence of 100 μM I942. Cell extracts were then prepared and immunoblotted with antibodies to SOCS3 protein, EPAC1 and GAPDH, as a loading control. Densitometry was then carried out on 3 western blots and results are shown as a histogram in the lower panel. Significant increases in SOCS3 protein expression, relative to diluent-stimulated control cells, are indicated; ***, p < 0.001. Significant inhibition of SOCS induction relative to I942-treated cells is also indicated; ###, p < 0.001.

c) HUVECs were preincubated for 30 min with the EPAC1 antagonist, ESI-09 (10 μM) or the broad-spectrum protein kinase A (PKA) inhibitor, H89 (10 μM), and then stimulated for a further 5 h with either 100 μM I942 or a combination of 10 μM forskolin and 10 μM rolipram (F/R) in the presence 10 μM MG132. Cell extracts were then prepared and immunoblotted with either the PKA substrate phospho-protein, CREB (pSer133), anti-SOCS3 and anti-GAPDH antibodies, respectively. Densitometry of SOCS3 expression from 3 separate experiments is shown in a histogram in the lower panel. Significant increases in SOCS3 expression relative to control cells are indicated, ***, p < 0.001, as are non-significant changes (ns).