Differential display and Northern blot analysis of the MADDAM cDNA fragment.

Differential Display (DD) of cDNA of different short-term cultured MOs (A) and Northern blot analysis of MADDAM expression in human blood MOs, lymphocytes, and mononuclear cells (B,C). MOs were stimulated for 4 hours with 1,25(OH)₂VD₃in the absence (2) or presence (4) of serum. Control cells were cultured for 4 hours without 1,25(OH)₂VD₃ under serum-free conditions (1) or with serum (3). RNA was extracted and cDNA amplified with the primer set AP1 and T₁₂MC. The cDNA fragment of MADDAM is marked (A). Total RNA of freshly isolated MOs (0 hours) or MOs cultured for 4 hours in the presence (serum) or absence (−) of serum with or without 10⁻⁷ mol/L 1,25(OH)₂VD₃ (VD) was isolated and Northern blot was performed according to “Materals and methods.” As a control for RNA loading, the membrane was rehybridized with an 18S rRNA-oligonucleotide and MADDAM signals were normalized to the corresponding 18S signals (B). In (C), the mRNA expression of MADDAM in mononuclear cells (MNCs), an elutriated lymphocyte fraction (LY), and elutriated MOs, stimulated for 4 hours with 10⁻⁷ mol/L VD under serum-free conditions, was compared by Northern blot analysis.