Figure 3

SOCS3 and SOCS1 participate to the induction of RAS activation and AKT phosphorylation in cytokine-activated keratinocytes. (a) RAS activity was measured on lysates obtained from MOCK (n=2), SOCS1 (n=4), SOCS2 (n=2) and SOCS3 (n=6) keratinocyte clones treated with TNF-α for 3 h. GTP-bound RAS was pulled-down using Raf-1 RBD agarose as substrate and detected by WB with an anti-RAS Ab. RAS activity was also analyzed in SOCS1- or SOCS3-interfered keratinocytes treated with IFN-γ/TNF-α for 12 or 24 h and compared with that observed in NC-transfected cells. Data are expressed as F.I.±S.D. between IFN-γ/TNF-α time-course points and untreated samples (time 0), to which a value of 1 were given. (b) Phosphorylation of AKT in Ser 473 was evaluated by immunoblotting on protein lysates obtained from MOCK and SOCS clones treated with TNF-α for 6 h, and from SOCS1-or SOCS3-silenced keratinocytes stimulated with IFN-γ/TNF-α for 18 or 36 h. Filters were stripped and re-probed with an anti-AKT Ab. In RNA interference experiments, F.I. values relative to SOCS1 or SOCS3-silenced keratinocytes were compared with those obtained from NC-interfered cells. In RNA interference experiments, data were obtained by three independent experiments. Values of P<0.05 (*) and P<0.01 (**) were considered significant