Western blot analysis of IGFBP5 expression in both cell lysate and conditioned medium. One million cells were cultured in 100-mm diameter tissue culture dish in the complete medium for 24 h followed by an additional 24-h incubation in the serum-free medium. Both cell lysate and conditioned medium were collected. The conditioned medium was concentrated 3 times using YM-10 column (Millipore) and 40 μg of protein from the cell lysate and 45 μl of the concentrated conditioned medium were subject for western blot analysis. The blot was re-probed with anti-actin to normalize for protein loading. wt, wild-type and mt, mutant form IGFBP5.