(A): Western blot analysis of Androgen receptor expression analysis (Upper Panel). Lower Panel: Expression of androgen receptor (AR) and E2A (E12/E47) bHLH transcription factor by RT-PCR. The gain of androgen receptor expression in DU145-Id4 cells as compared to DU145 cells at the transcript and protein level is evident. The total RNA and protein was purified from DU145-Id4 at passage 28 and analyzed for the expression studies. As controls, the parental, mock transfected DU145 cells (AR -ve) and LNCaP (AR +ve) cell lines were used. The expression of beta-actin was used as loading and RT-PCR control. The data is representative of at least three different RT-PCR reactions and Western blot analysis. The following primers were used: E2A F-5' CAC CAG CCC TCA TGC ACA ACC, R-5' CTC CAA CCA CAC CTG ACA C and androgen receptor (AR): F-5' ATG GTG AGC AGA GTG CCC TA and R-5' GTG GTG CTG GAA GCC TCT CCT. (B) Real time PCR analysis, performed on the same batch of reverse transcribed RNA used in panel A confirms the RT-PCR data. The fold change in AR expression is normalized to beta-actin (3 different RT reactions) and calculated by the Δ Ct method as described in materials and methods section (*** P < 0.001).