Fig. 1. Analysis of constitutive B DNA-binding in human primary prostate cells and PC cell lines. (A) EMSA analysis of B-binding. PrEC, normal epithelial prostate primary cultures; LNCaP and MDA PC 2b are androgen-dependent cell lines; CL2 (derived from LNCaP cells), JCA1, PC3 and DU145 are androgen-independent PC cell lines. Nuclear proteins (10 µg/reaction) from untreated cells and LNCaP cells treated with TNF- were used for electrophoretic mobility shift assay (EMSA). Composition of dimers is indicated. Data are shown for one representative experiment. (B) Identification of nuclear B-binding complexes by EMSSA. Nuclear proteins from PC3 cells were incubated with a labeled B oligonucleotide and antibodies against p50 and p65 proteins. DNA binding activity was analyzed by EMSA. Composition of dimers is indicated. (C) EMSA analysis of Sp1-binding. Nuclear proteins (10 µg/reaction) from the same cells as in Fig. 1A, were used for EMSA with Sp1 oligonucleotide. Composition of dimers is indicated. Data are shown for one representative experiment.