Figure 1

Hoechst-IGF1 synthesis and bioactivity.

(A) Hoechst-amine was functionalized by the addition of a PEG-Biotin to allow for conjugation with IGF-1. (B) Using the biotin-streptavidin interaction, Hoechst-biotin was incubated with biotinylated-IGF-1 and tetravalent streptavidin to create the Hoechst-IGF1 (H-IGF1) complex. A control compound, streptavidin-IGF1 (S-IGF1) containing only streptavidin, biotin and biotinylated-IGF-1 was also assembled. (C) Immunoblot for IGF-1 demonstrating the size of H-IGF1 species at 70 and 80 kDa (right lane) compared to biotinylated-IGF-1 at 10 kDa (left lane). (D) Live and methanol fixed RAW 264.7 macrophages were treated with H-IGF1 or Hoechst-biotin for 15 minutes. Hoechst compounds remained membrane impermeable to live cells as demonstrated by low levels of blue fluorescence. In contrast, permeabilized cells had high levels of blue, nuclear fluorescence demonstrating the DNA binding capability of Hoechst. White scale bar represents 100 μm. (E) Cardiomyoblasts (H9c2 cells) were treated with control media, streptavidin, IGF-1, biotinylated-IGF-1 or H-IGF1 for 15 minutes. Cells were homogenized and immunoblotted for Akt phosphorylation. H-IGF1 treatment activated Akt to a similar extent as IGF-1 and biotinylated-IGF-1.