Figure 3.

Bard1 expression in different Bard1-antisense and ribozyme expressing cell lines. (A) 10 μg of total RNA extracted from TAC-2 cell lines were tested by RNase protection assays with probes against Bard1 and GAPDH (panel a) against Atm (panel b), and against p21 (panel c). TAC-2, nontransfected cells; PC3, mock-transfected PC3; Bard1-sense, control transfected Bard1-sense cells; AB-heterog, parental anti-Bard1 transfected cells; AB-H to AB-L, clonal cell lines derived from anti-Bard1; RB-18, ribozyme-transfected cells. Arrows, antisense protected band of correct size, additional slower migrating bands were seen in some samples after longer exposure. (B) Western blot analysis of protein levels in antisense and ribozyme expressing cells. Antibodies generated against the Bard1 peptides PVC, WFS, and MIQ were tested on Western blots of protein extracts from TAC-2 cells and mock-transfected PC3 cells (PC3) cells (panel a). Preimmune serum (P), corresponding to PVC antiserum, and purified PVC (1), WFS (2), and MIQ (3) antibodies were probed in a 1:100 dilution. Protein extracts from mock-transfected TAC-2 cells (PC3), ribozyme-expressing cells (RB-18), and antisense expressing cells (AB-H, AB-I, and AB-K) were blotted onto nitrocellulose filters and detected with PVC anti-Bard1 antibodies by chemiluminescence (panel b).