FIGURE 4.

Differences in IL-23-mediated STAT3 activation in HeLa and Ba/F3-gp130 cells. A, IL-23R variants containing two or three mutations were generated by site-directed mutagenesis to define STAT3 recruitment sites within the cytoplasmic domain. B, p409 expression vectors containing the cDNAs as indicated were co-transfected into HeLa cells, and analysis was performed as described in the legend to Fig. 3B. Respective stably transduced Ba/F3-gp130 cells were generated as described and analyzed as mentioned. The presented Western blots originate from different membranes and are therefore separated by black lines. Data are representative of at least two independent experiments. C, proliferation of stably transduced Ba/F3 cells based on the colorimetric CellTiter-Blue® Cell viability assay was performed as described. Values represent the mean value of three repetitions and were normalized. For comparison of the independent stably transduced Ba/F3-gp130 cell lines, n-fold proliferation was calculated by setting the negative control of each Ba/F3 cell line to a value of 1. Data are representative of at least two independent experiments. D, IL-23R variants containing mutations of mouse Tyr-626 were washed three times with PBS and starved for 2 h in serum-free DMEM. 1 × 10⁶ cells were stimulated for 30 min with 0.2% HIL-6 or HIL-23, harvested by centrifugation, fixed in 2% (w/v) paraformaldehyde, and permeabilized in 90% (v/v) methanol. Cells were stained for phospho-STAT3 and STAT3 overnight and analyzed by flow cytometry. E, stably transduced Ba/F3 cells were washed three times, starved for 4 h, and stimulated with 0.2% HIL-23 for 60 min. Cellular lysates were prepared, and equal amounts of proteins (50 μg/lane) were loaded onto SDS gels. Western blots were performed using antibodies specific for phospho-STAT1 and STAT1. Western blot data are shown for one representative experiment. Error bars, S.D.