Fig. 3.

c-Jun is required, but not sufficient, to promote SOCS3 induction by cyclic AMP. (a) HUVECs were transfected with vectors expressing human c-Jun or c-Jun where Ser63 and Ser73 had been converted to alanine (Ser63/73Ala). Cells were then stimulated with F/R (5 h) in the presence or absence of the proteasome inhibitor MG132 to stabilise SOCS3 expression. Cell extracts were then immunoblotted with the indicated antibodies and denstitometric values are displayed as a histogram in the lower panel. Significant increases in SOCS3 immunoreactivity relative to diluent-treated cells are shown, ^∗P < 0.05 and ^∗∗∗P < 0.001, as is the significant in increase in immunoreactivity in F/R + MG132 cells relative to cells treated with MG132 alone (^###P < 0.001, n = 3). (b) HUVECs were transfected with wild type human c-Jun or mutant c-Jun (Ser63/73Ala), and then stimulated with combinations of F/R and IL6 for 5 h as indicated. Cell extracts were then immunoblotted with antibodies that recognise c-Jun, total STAT3 and STAT3 phosphorylated on Tyr705. Densitometric analysis of immunoblots was carried out and results are presented as a histogram in the lower panel. Significant increases in pSTAT3 (Tyr 705) immunoreactivity relative to diluent-treated cells are shown, ^∗∗∗P < 0.001, as are significant decreases in immunoreactivity in c-Jun transfected cells, relative to mock-transfected cells (^###P < 0.001, n = 3). (c) Wild type or c-Jun knockout mouse embryonic fibroblasts (MEFs) were transfected with or without (mock) c-Jun or c-Jun (Ser63/73Ala) and then stimulated in the presence or absence of F/R (5 h) plus MG132, as indicted. Cells extracts were then prepared and immunoblotted with the indicated antibodies. Densitometric values from immunoblots are displayed in the lower panel where significant increases in SOCS3 immunoreactivity relative to diluent-treated cells are shown, ^∗∗∗P < 0.001 (n = 3).