fl-hKITL regulates the expression of GRα in human EBs. (A) Quantitative RT-PCR of the levels of GRα mRNA expressed by day 10 EBs generated in HEMA cultures stimulated with either hKITL or mKITL, as indicated. The number of independent experiments included in the analyses (each one with a different donor) is indicated by n. mRNA levels are presented in relative units calculated using the average 2−ΔCt observed with EBs generated with fl-hKITL as 1. (B) WB analysis for GRα protein content of day 10 EBs generated in the presence of fl-hKITL, tr-hKITL, and mKITL, as indicated. Two representative experiments, 1 with CB-derived and 1 with AB-derived EBs are presented. GAPDH was analyzed as loading control. For further details see legend of Fig. 7. (C) Levels of STAT5 phosphorylation expressed by day 10 EBs obtained in HEMA cultures stimulated with either fl-hKITL, tr-hKITL, or mKITL and then exposed or not to DXM for 15 min. (D) Quantitative RT-PCR and (E) western blot analysis of the levels of GRα mRNA and protein expressed by day 10 EBs generated in HEMA cultures stimulated with fl-hKITL [shaded area in (C) and Prol in (D)], GFD for 4 h (time 0), and then exposed to either fl-hKITL, tr-hKITL, or mKITL (50 ng/mL in all cases) for up to 4 h as indicated. mRNA and protein levels were analyzed on the same cells. mRNA levels are expressed in relative units using 2−ΔCt obtained with GFD EBs as 1 and presented as mean (±SD) of 3 separate determinations. Asterisk (*) indicates <0.01 with respect to the corresponding values observed with fl-hKITL. (F) Western blot analysis of the levels of GRα, phospho-extracellular signal regulated kinases (P-ERK), and total ERK 1/2 and GAPDH (as loading control) in day 10 EBs generated in the presence of fl-hKITL, and exposed to GFD for 4 h. In some of the samples, the last hour of GFD occurred in the presence of U0126 (100 μM). GFD with or without U0126 EBs was then exposed to fl-hKITL for 15 min and 1 h, as indicated. The FC of the various bands with respect to the levels of GAPDH is also indicated. Similar results were observed in a separate experiment. GR, glucocorticoid receptor; RT-PCR, real-time polymerase chain reaction; AB, adult blood.