Figure 4.

Down-regulation of BI-1 expression in PC-3, LNCaP, and DU-145 cells transfected with sequence-specific duplex siRNA oligonucleotides (D-BI-1) against the BI-1 gene. As negative controls, single-strand BI-1 sense and antisense oligonucleotides, duplex siRNA oligonucleotides against the firefly luciferase gene and the Mat-8 gene were used for transfection experiments (control). After transfection both attached and floating PC-3, LNCaP, and DU-145 cells were collected and used for both RNA and protein isolation, respectively. A: Northern blot analysis of total RNA (5 μg) derived from siRNA-transfected (D-BI-1) and control-transfected PC-3, LNCaP and DU-145 cells was performed using a human BI-1 cDNA fragment as a probe. Rehybridization of the same filter was carried out with a cDNA probe for β-actin. B: The mRNA signals were scanned using the phosphorimager Molecular Imager FX and the difference in the expression of BI-1 transcripts was calculated relative to the β-actin standards. Results of the analysis for BI-1 are shown in the bar graph. The labels for the bars correspond to those used in A. C: Western blot analysis of transfected PC-3, LNCaP, and DU-145 cells using BI-1 (top panel) or α-tubulin-specific (bottom panel) antibodies. The Western blot was stripped and re-probed with an α-tubulin antibody to check for equal loading (50 μg) of total protein.