Analysis of retinoic acid response element (RARE)–luciferase reporter gene activation and STAT3 phosphorylation in EPRO-Gr inductions. Induced EPRO-Gr cells were analyzed for activation of RAREs (A,B) by transient transfections with pBStkRARE-luc and treatment of cells with various induction media as outlined in Figure 2 (u-uninduced; a-ATRA; g-GCSF; g/a-GCSF plus ATRA) and for activation of STAT3 (C) under the same conditions. (A) Shown is a diagram of the pBStkRARE luciferase reporter construct containing 3 RAREs placed in series upstream of the 158-bp tk promoter fragment and luciferase (“Materials and methods”). (B) Cell lysates were obtained 24 hours after transfection, and luciferase activity was measured. A beta-galactosidase expression vector was cotransfected at a ratio of 20:1 to normalize for transfection efficiency. Luciferase activity in EPRO-Gr cells is reported as fold increase in relative light units (RLU) above pBStkRARE-luc in control medium (GM-SCF). The figure represents the mean ± SE value from 3 independent experiments, each performed in duplicate. (C) Following 60 minutes of serum starvation, total cell lysates (5 × 10³ cells/μL2 × GSB) were harvested from uninduced (u) EPRO-Gr cells, and cells induced with ATRA (a), G-CSF (g), and G-CSF plus ATRA (g/a) for 15 minutes. Lysates were subjected to 4%-12% SDS-PAGE. Western analysis was subsequently performed and probed sequentially with rabbit-polyclonal IgG anti–phospho-STAT3 (Tyr 705) and rabbit-polyclonal IgG anti-STAT3 as primary antibodies. Binding of primary antibodies was detected with HRP-linked anti–rabbit IgG antibodies and chemiluminescent detection. As controls, equivalent amounts of lysates from HeLa cells treated with INF-α (positive control; 100 ng/mL) and untreated HeLa cells (negative control) were run in parallel.