Sp1 positively regulates the CD99 promoter in

D melanogaster SL2 cells and in 293T. (A) Activation of the CD99 promoter by Sp1 in D melanogaster SL2 cells. To achieve sufficient protein expression in transient transfection assays, various amounts of Drosophila β-actin promoter-driven Sp1 (pAcSp1) were used. One microgram wild-type promoter construct p(−317/+123)luc (░) or the construct containing mutated Sp1 recognition sites in the promoter, p123mGC (■), was transfected into SL2 cells. (B) Effect of the activation-domain deletion mutant of Sp1 on the CD99 promoter activation. A construct containing either 327 or 168 carboxyl-terminal amino acid residues of Sp1, pPac327C (▨) and pPac168C (▪), respectively, was cotransfected with the wild-type CD99 promoter construct, p(−317/+123)luc. (C) Effects of Sp1 and Sp3 on the CD99 promoter in 293T. The wild-type promoter-luciferase fusion construct, p(−317/+123)luc was cotransfected with expression plasmid for Sp1 or Sp3 into 293T cells. The internal control, 1 μg pBip670CAT, was cotransfected. Transfected cells were prepared in 2 separate ways for luciferase and CAT assay and for Western blot analysis. For luciferase and CAT assay, cells were prepared with passive lysis buffer solution. For Western blot analysis, cells were washed and solubilized in lysis buffer as described in “Materials and methods.” After protein quantitation, the lysates were separated by 10% SDS PAGE and electroblotted onto the nitrocellulose filters. The same filter was hybridized with monoclonal antibodies such as D-20, 1C6, and anti-Calnexin for Sp1, Sp3, and Calnexin, respectively. Relative luciferase activity is expressed as relative activity over the negative control transfection with pcDNA3.