Fig. 6

High IL-6Rα/gp130 receptor ratio in HepG2-IL-6Rα cells ablates difference between trans- and classic signalling. a Expression of gp130 and IL-6Rα on the surface of HepG2-IL-6Rα cells was quantified by flow cytometry using QIFIKIT. Mean ± STD values from n = 4 independent experiments are shown. Expression of STAT3 in HepG2-IL-6Rα cells was quantified by Western blotting using recombinant calibrator proteins. Mean ± STD values from n = 7 independent experiments are shown. b HepG2-IL-6Rα cells were stimulated with IL-6 (blue) and Hy-IL-6 (red) (0.17 nM). STAT3 phosphorylation and expression of STAT3, SOCS3, and HSC70 protein were evaluated by Western blotting. Expression of STAT3 and HSC70 served as loading control. The expression of SOCS3 mRNA was analysed using qRT-PCR. Diamonds correspond to the mean and bars to the STD of n = 4 experiments. Data normalization was performed as described in Additional file 1: Text S3. c HepG2-IL-6Rα cells were stimulated with indicated amounts of IL-6 (blue bars) or Hy-IL-6 (red bars). STAT3 phosphorylation was evaluated by intracellular flow cytometry using specific fluorescent antibodies against STAT3 (p)Y705. For independent experiments mean fluorescence of 10⁴ cells per cytokine concentration was calculated. Data are given as mean ± STD from n = 3 experiments. d HepG2-IL-6Rα cells were pretreated with cycloheximide for 30 min and subsequently stimulated with 0.17 nM IL-6 (blue) and Hy-IL-6 (red), respectively. STAT3 phosphorylation and STAT3 expression were evaluated by Western blotting. STAT3 expression served as loading control. The expression of SOCS3 mRNA was analysed using qRT-PCR. Diamonds correspond to the mean and bars to the standard deviation for n = 3 independent experiments. Data normalization was performed as described in Additional file 1: Text S3