Confocal and biochemical analysis of siRNA-mediated knockdown of BRCA1 protein in HeLa cells. A: Cells transfected with plasmid pcDNA3.1 GFP show punctate nuclear staining for BRCA1 protein (green), and for nucleolin (red), which is predominantly colocalized (yellow). Original magnification ×630. B: Cells transfected with the pSUPER GFP plasmid show punctate nuclear staining, in which BRCA1 (green) staining is sometimes, but not always colocalized with nucleolin (yellow). Original magnification ×630. C: BRCA1 protein staining was diminished in cells transfected with the pSUPER HBRCA1 plasmid; counterstained with DAPI. Original magnification ×630. D: The 220-kDa BRCA1 protein is significantly knocked down in whole cell lysates from HeLa cells transfected with pSUPER HBRCA1 plasmid (lane 3); whereas the 220-kDa BRCA1 protein is clearly visible in control cells transfected with empty pSUPER plasmid (lane 1), or pSUPER GFP plasmid (lane 2). The loading control α-tubulin, shows equivalent concentrations in the lysates, and is not diminished in the siRNA-HBRCA1-treated cells.