Figure 5. JAK3 inhibition affects the phosphorylation levels and DNA binding activity of NF-κB and CREB in monocytes.

Purified human monocytes were pretreated with JAK3 inhibitor (T-1377, 10 nM), control peptide, or NF-κB inhibitory peptide and then stimulated with LPS. Whole cell lysates, cytosoplasmic and nuclear cell fractions were collected after stimulation with LPS at the time points indicated. (A) JAK3 inhibition increased the phosphorylation of NF-κB (S536) upon LPS stimulation. (B) Densitometer scans of phospho-NF-κB (S536) and total β-actin were performed and recorded as the ratio of phospho-NF-κB (S536): total β-actin. NF-κB and CREB DNA binding assays were performed to measure the levels of total NF-κBp65 (C) (10 μg nuclear lysate) and CREB (S133) (D) (20 μg nuclear lysate) in monocytes stimulated with LPS for 4 h in the presence and absence of the JAK3 inhibitor (T-1377), GSK3 inhibitor (SB216763) or both. For E and F, the functional effect of JAK3 inhibition on NF-κB P65 activity in LPS stimulated monocytes was assessed by pre-treatment of cells with a control peptide or NF-κB inhibitor peptide (100 μM) followed by stimulation with LPS for 20 h; The levels of IL-12P40 (E) and IL-10 (F) were determined by ELISA. For C, D, E, and F, data represents the arithmetic mean ± S.D. of three biological replicates. * and *** indicates statistically significant at P<0.05 and P<0.001, respectively.