STAT3 is constitutively activated in human macrophages and is suppressed by NaSal treatment. (A-B) Primary human macrophages were treated with control medium or NaSal (20 mM), where indicated, for 48 hours. Cells were harvested and nuclear proteins were extracted and used for the EMSA. The radiolabeled probe employed represents the murine Mcl-1 SIE (5′-TTTCCCCTTTTACGGGAAGTCCT-3′).^8,11  Added to the reaction mixture to define the specificity of the bound proteins were 100-fold unlabeled probe of murine Mcl-1 (Mcl-1) (A) and antibodies to STAT3 (ST3) or STAT1 (ST1) and control antibody (Ctrl) (B). (C) The STAT3 is constitutively phosphorylated at the Ser727 site and not the Tyr705 site in primary human macrophages. Nuclear proteins (30 μg) extracted from primary human macrophages or U266 cells were employed to detect phospho-Ser727 and phospho-Tyr705 STAT3 by Western blot analysis employing monospecific antibodies. (D) NaSal treatment inhibits the constitutive Ser⁷²⁷ phosphorylation of STAT3 in human macrophages. Macrophages were treated with NaSal (20 mM) for 48 hours. Cell lysates were collected and Western blot assays were conducted using indicated antibodies to phospho-serine STAT3, total STAT3, and tubulin. Nuclear proteins also were used to detect the effect of NaSal on phospho-serine STAT3, where indicated. (E) Akt-1 does not regulate STAT3 phosphorylation in human macrophages. Human macrophages were infected with adenoviral vectors (100 moi) expressing the control or activated Akt-1 (MyrAkt-1) for 24 hours. Cell lysates were collected and Western blot assays were conducted using indicated antibodies to phospho-serine STAT3 and tubulin.