NFAT protein occupies the distal NFAT site, and calcium mobilization is sufficient to induce endogenous CD154 expression. (A) Nuclear extracts prepared from untreated and PMA differentiated CMK cells were incubated with an oligonucleotide probe representing the −340 to −300 nt region of the CD154 promoter (the distal NFAT site in bold). Specificity of the protein complex formation was demonstrated by inclusion of cold oligonucleotides in the binding reaction. Specific competitor (spec com) contained a 50-fold molar excess of the identical probe. The nonspecific competitor reaction contains 50-fold molar excess of the identical probe with 2 point mutations in the distal NF-AT site (GGAAA to GAGAA). Supershifting is observed in reactions containing anti-NFATc and anti-NFATp but not immunoglobulin control (IgCon). (B) Relative CD154 mRNA expression in PMA-differentiated CMK cells and primary murine megakaryocytes (CD41+) derived from 12-day cytokine-differentiated LSK cells in the presence or absence of CsA. (C) CD154 mRNA expression in undifferentiated and differentiated mouse primary cells (LSK and CD41+), CMK, and K562 cells left untreated or treated with PMA, ionomycin, or both (or both PMA and ionomycin in the presence of CsA) for 2 hours. The bars represent means plus or minus the standard error from 2 independent experiments.