(A) Id4 expression is generally absent in DU145 cells but present in androgen receptor positive LNCaP cells. Treatment of DU145 cells with global de-methylation agent 5'-Aza-2-Deoxycytidine (5-AZA-CdR) leads to Id4 expression as determined by western blot analysis (upper panel) and reverse transcriptase polymerase chain reaction (RT-PCR, lower panel). Beta-actin was used as a loading and RT-PCR control. The primer pairs used for amplification were described in . (B) Analysis of Id4 methylation in human fibroblasts (BJ) and the prostate cancer cell lines DU145, DU145-Id4 (DU145 cells with constitutively expressed Id4) and LNCaP by methylation specific PCR (MSP). The presence of a PCR band in lanes marked "M" indicates a methylated gene sequence, the presence of a PCR band in lanes marked "U" indicates an un-methylated gene sequence. Normal peripheral blood cells (N), in vitro methylated DNA (IVD) and water served as controls.