Figure 3. Interaction of IHCA gp130 mutants with wild-type gp130 and OSMR. (A) MYC- or Flag-tagged constructs expressing either wild-type (WT) gp130 or the inflammatory hepatocellular adenoma (IHCA)-associated gp130 mutants A418 or Y186 were co-transfected (1:1) into Hep3B cells. Gp130 hetero- and homo-dimerization in the absence of interleukin-6 (IL-6) was assessed by immunoprecipitation using the anti-Flag antibodies followed by immunoblotting. (B and C) Hep3B cells (n = 3) were co-transfected with a control vector or a plasmid expressing the Y186 gp130 mutant and a control siRNA (−) or a siRNA specific for WT gp130 (+) and quantitative PCR was used to examine the effects on transcription of STAT3 target genes. Shown is the mean ± SD expression level of endogenous CRP (B) and SOCS3 (C) transcripts in cell expressing gp130 mutants relative to WT gp130 (1-fold). On hundred ng/mL IL-6 was added to the culture medium where indicated. (D) The dimerization potential of the oncostatin M receptor (OSMR) with WT or mutant gp130 in the absence of OSM was assessed in Hep3B cells expressing Flag-tagged WT or mutant gp130. Shown are the immunoblots of immunoprecipitates obtained with anti-OSMR antibodies in the presence or in the absence of 100 ng/mL OSM. (E) Hep3B cells were co-transfected with the STAT3-luciferase reporter and a control vector (EP) or plasmids encoding the Y186 gp130 mutant or OSMR, as indicated. One hundred ng/mL OSM was added when indicated. Shown are the mean ± SD luciferase activities determined from triplicate co-transfections. (F) The knockdown of OSMR in Hep3B cells (n = 3) impairs STAT3 activity a driven by the Y186 gp130 mutant or treatment with 100 ng/mL OSM. Shown are the mean luciferase activities ± SD relative to cells transfected with the EP only. Results were confirmed using a second siRNA (data not shown). Statistical significance was determined by 2-tailed Student t test; **P < 0.01; ***P < 0.001.