Figure 2

IL-4-activated JAK signaling results in the phosphorylation of STATs and Akt. Lymphocytes were preincubated for 30 min prior to the addition of IL-4 with/without pyridon 6. After 2 h IL-4 stimulation, activation of JAK downstream signaling molecules (others than STAT6) was investigated in cell lysates using Western Blotting followed by densitometry. Representative immunoblots are shown; the same blot was probed sequentially with different antibodies. Densitometry results are given as mean % expression of loading control ± SEM in the bar graphs; statistical analysis was performed using the Friedman and Dunn’s test; *P < 0.05. A) Phosphorylation of STAT1 (Tyrosine 701, 84/91 kDa) and STAT3 (Tyrosine 705, 79/86 kD and Serine 727, 86 kDa) and inhibition by pyridon 6; loading was controlled by staining for non-phosphorylated STAT3 (79/86 kDa). The bar graphs show the mean expression of phosphorylated STAT1 (n = 3 independent experiments), Tyrosine-phosphorylated STAT3 (n = 6 independent experiments, and Serine-phosphorylated STAT3 (n = 3 independent experiments) in % of STAT3, respectively. B) Phosphorylation of STAT5 (Tyrosine 694, 90 kDa) and of Akt (Serine 473, 60 kDa) and inhibition by pyridon 6; loading was controlled by staining for beta-actin (42 kDa). The bar graphs show the mean expression of phospho-STAT5 (n = 3 independent experiments) and phospho-Akt (n = 3 independent experiments) in % of beta-actin, respectively. C) Acetylation of STAT3 (Lysine 685, 79/86 kDa) and inhibition by pyridon 6; loading was controlled by staining for STAT3 (79/86 kDa). The bar graph shows the expression of Acetyl-STAT3 (n = 3 independent experiments) in % of STAT3. Ac; acetylated; Lys; Lysine; Ser, Serine; Tyr, Tyrosine.