Fig. 2

TGF-β induces BARD1β overexpression and localization. a Schematic representation of cDNA structure of FL BARD1 and N-terminally truncated isoform BARD1β. Approximate locations of protein motifs RING finger (RING), ankyrin repeats (ANK), and BRCT domains (BRCT) of BARD1 are indicated. Green exons indicate protein coding open reading frame (ORF). BARD1β encodes an alternative ORF in the first exon. Arrows indicated approximate positions of epitopes reactive with the antibodies used. b Lung epithelial cells (A549) and fibroblasts (L929) were cultured in absence (NT) or presence of TGF-β1, and cell extracts were prepared after 24 and 48 h. Western blots of cell extracts were probed with anti-BARD1 BL (exon 4), BARD1β-specific P25 (alternative ORF of BARD1β), α-SMA, E-cadherin, fibronectin, and β-actin antibodies. c A549 were treated with TGF-β1 and cells were fixed after 48 h. Immunofluoresence was performed with anti-E-cadherin, fibronectin, or BARD1 BL (exon 4) antibodies. d Co-immunostaining with anti-BARD1 BL and fibronectin (Fib) antibodies. TGF-β1 modulates BARD1 localization to areas at the cell membrane (yellow arrows) and cytoplasmic vesicles (white arrows), similar to and co-staining with fibronectin. Scale bars 20 μm