Figure 2

Suppression of endogenous AR expression in LNCaP cells using the pSiAR-EGFP expression construct. LNCaP cells were transfected with either the pSiAR-EGFP or the pSiAR-EGFP construct using the Lipofectemine 2000 protocol. (A) Immunocytochemical detection of AR expression in transfected cells. To determine AR expression in LNCaP cells, following transfection, cells were stained with mouse anti-human AR monoclonal antibody (1:100 dilution) followed by AlexaR FluorR 594 conjugated goat anti-mouse IgG secondary antibody (2 mg/ml) incubation. The AR staining was detected by fluorescent microscopy (BX51, Olympus). The images were composite between the red with the positive in AR staining with the phase-contract from the same field. Suppression of endogenous AR expression was demonstrated by the absence of red fluorescence staining as indicated by arrows. DAPI staining showed the number of cells in the same field. (B) Western blot analysis of AR expression in pSiAR-EGFP and control transfected cells. At 7 days following transfection, both GFP-positive and GFP-negative cells were collected through cell sorter; and cells were lysed to prepare cellular protein extracts. Aliquots of 20 μg total cellular protein were loaded into Tris-HCl gels and transferred to PDVF membranes. The AR expression was determined by incubating with mouse anti-human AR monoclonal antibody (1:500) followed by the HRP-conjugated anti-mouse IgG (1:125,000) secondary antibody incubation. Immunoreactive signals were detected using ECL. Levels of β-actin expression was also determined in each sample and used as protein loading control.