Fig. 2

A–L: D427H mutation does not alter tyrosine phosphorylation of STAT3 upon cytokine stimulation. U3A and HeLa cells were transfected with GFP/SNAP-tagged variants of STAT3 and subsequently stimulated with different cytokines for the indicated times. A Representative immunoblot of whole cell extracts from U3A cells expressing GFP-tagged STAT3 proteins after treatment for 0, 15, and 30 min with 25 ng/ml of recombinant IL-6 and the quantification thereof from three independent transfection experiments (B). Similar experiments were performed with GFP-tagged STAT3 variants after 50 ng/ml IFNγ stimulation as shown in the representative immunoblot (C) and the quantification thereof from three independent transfection experiments (D). E Western blot of whole cell extracts from U3A cells expressing SNAP-tagged STAT3 variants after treatment for 0, 15, and 30 min with recombinant IL-6 and the quantification thereof from three independent transfection experiments (F). G Representative immunoblot of whole cell extracts from U3A cells expressing SNAP-tagged STAT3 mutants after treatment for 0, 15, and 30 min with recombinant IFNγ and the quantification thereof from three independent transfection experiments (H). I–L Western-blotting experiments demonstrating unchanged kinetics of tyrosine phosphorylation of STAT3-D427H as compared to WT and the positive control F174A performed in HeLa cells expressing the STAT3 variants which were subsequently stimulated with 50 ng/ml of IFNγ for the indicated times. I Representative immunoblot of whole cell extracts from HeLa cells expressing GFP-tagged STAT3 mutants after IFNγ stimulation and the quantification thereof from three independent transfection experiments (J). Similar experiments were performed with SNAP-tagged STAT3 variants as shown in a representative immunoblot (K) and the quantification thereof from three independent transfection experiments (L). Means ± standard deviations from three independent experiments with *p ≤ 0.05. Arrows indicate the respective STAT3-GFP or STAT3-SNAP bands of interest