Figure 3.

Expression of genes associated with androgen independence is decreased in C81 EBP1 transfectants. A, lysates of logarithmically growing clones of C81 EBP1 transfectants (E1 and E2) or vector controls (V1 and V2) were resolved by SDS-PAGE and immunoblotted for AR, BCL-2, BAD, FLAG, EBP1, and actin as indicated. Representative of three experiments. B, secreted PSA as measured by ELISA. EBP1 and vector control cells were plated at 1 × 10 ⁶ per 100 mm dish in complete medium. Forty-eight hours later, conditioned medium was collected and frozen. PSA activity was assessed by ELISA. Levels of PSA were adjusted to total cell number. Representative of two experiments. C, validation of mRNA levels in EBP1 transfectants. Quantitative reverse transcription-PCR was done for the kallikrein-2, AGR2, and TSPY genes in EBP1 and vector transfectants. The relative levels of all test mRNAs were normalized to β-actin. Representative of three experiments using different sets of cells. D, expression of AGR2 protein in vector and EBP1 C81 transfectants. Lysates of vector control or EBP1-transfected cells grown in complete medium were resolved by SDS-PAGE and analyzed by Western blotting with antibody to AGR2.