Figure 2

Phosphorylation state and signaling activity of CAgp130. T-REx-293-WTgp130-YFP and T-REx-293-CAgp130-YFP were left untreated or expression was induced with 0.5 μg/ml (A) or 20 ng/ml (B, C and D) dox for 24 h. Cells were stimulated with 200 U/ml IL-6 and 0.5 μg/ml sIL-6Rα for 15 min (A), 30 min (B and D) or for the indicated periods of time (C) or left unstimulated. In (C) cells were puls-stimulated and the stimulus was removed after 15 min of incubation. (A) Gp130 was immunoprecipitated from TCLs using an antibody against the C-terminus of gp130. Precipitates were analyzed by immunoblotting using Abs against pTyr and gp130. Asterisks mark phosphorylation signal of endogenous gp130. Black and grey arrows mark the high and low glycosylated form of WTgp130-YFP and CAgp130-YFP respectively. (B) Activation of the JAK/Stat pathway was analyzed by immunoblotting of TCLs with Abs against pStat3(Y705), pStat3(S727), pStat1(Y701), Stat3, Stat1, gp130 and actin as loading control. (C) TCLs of depicted cells were analyzed by immunoblotting using Abs against pStat3(Y705), Stat3, gp130, SOCS3 and actin as loading control. For the SOCS3 positive control HEK293 cells were transiently transfected with a SOCS3 encoding plasmid. (D) Activation of the JAK/Erk pathway was analyzed by immunoblotting of TCLs with Abs against pSHP2, pErk1/2, SHP2, Erk1/2 and gp130.