FIGURE 6.
Tyrosine phenylalanine substitutions resulted in altered kinetics of STAT3 activation. A, two independent stable transduced Ba/F3-gp130 cell lines (36 and 38) containing the IL-12Rβ1 and mutated IL-23R (Y426F/Y504F/Y542F/Y626F) cDNA were starved in serum-free medium and subsequently stimulated with 0.2% HIL-23 for 10 min. Cells were harvested, and whole cell lysates were prepared and analyzed by Western blot using antibodies specific against phospho-STAT3 and STAT3. B, STAT3 activation was evaluated by intracellular staining. Ba/F3-gp130 cells and the FFFF Ba/F3 cell line containing mutated IL-23R (Y426F/Y504F/Y542F/Y626F) were washed three times with PBS and starved in serum-free DMEM. 1 × 106 cells were stimulated for the times indicated with 0.2% HIL-23, harvested by centrifugation, fixed in 2% (w/v) paraformaldehyde, permeabilized in 90% (v/v) methanol, stained, and analyzed by flow cytometry.