hTERT promoter occupancy in vivo by chromatin immunoprecipitation assay. Control PBMCs, wild-type Tax-transduced PBMCs, and mutant Tax (G148V)–transduced PBMCs were cultured in RPMI 1640 supplemented with 20% FBS and 50 U/mL IL-2 for 8 weeks and used as described in “Materials and methods.” The HTLV-I–transformed cell line MT-2 was included. The following antibodies (all from Upstate Biotechnology) were used for immunoprecipitation: anti-Sp1 (sc-59), anti-p53 (sc-126), anti–c-Myc (sc-764), anti-Max (sc-197), and anti–NF-κB p65 (sc-109). Samples with no antibody (No Ab) as negative controls and samples corresponding to 1% or 10% input as loading controls were also PCR amplified. PCR products were run on a 10% polyacrylamide gel and stained with SYBR Green I. (A) The representative results of in vivo protein binding to the hTERT promoter. For NF-κB p65, PCR products from the IL-8 promoter region as a positive control are also shown. (B) Quantitative analysis of the binding of Sp1 (B) and c-Myc (C) proteins to the hTERT promoter. The PCR products from 1% input samples were used to normalize the Sp1 and c-Myc binding in each cell line. The value of PBMCs is defined as 1.0 and those of the other 3 cell lines are expressed relatively. The data (average ± SD) are from 3 independent experiments.