Establishment of the wild-type and mutant forms of IGFBP5 stably expressing cell lines. A. Schematic diagram of the IGFBP5 cDNA fragments used to generate the IGFBP5 expression constructs. Wild-type IGFBP5 contains a NLS located at the C-terminal domain of the protein from amino acid 201 to 218 and indicated by a gray solid square. For the mutant form of IGFBP5, 5 amino acids in the NLS from 214–218 (black characters) were deleted. B. Western blot analysis of IGFBP5 expression. MBA-MD-435 breast cancer cells were stably transfected with either the wild-type or the mutant forms of IGFBP5 construct and the whole cell lysate from parental MBA-435 cells (P) and different stable lines were subjected to western blot analysis (40 μg/lane) using an anti-human IGFBP5 antibody. The blot was re-probed with actin to normalize for protein loading. Neither parental nor vector-transfectant (V) cells had detectable levels of IGFBP5. 10 ng of purchased human recombinant IGFBP5 protein was served as a positive control (+). wt, wild-type and d, deletion form IGFBP5.