Figure 4

Pull down experiments using CBD peptides. 20 mM biotinylated peptide encompassing either the putative binding domain within MA or a consensus binding motif were inoculated with 50 μl NIH3T3 cell lysate for 90minutes (from 2 ml lysate of semiconfluent T75 culture flask). Complexes were immobilized using 10 μl streptavidin coated paramagnetic microbeads and μ column (Miltenyi). Washed samples were eluted and 15 μl of 80 μl eluate were separated by SDS-PAGE, blotted to PVDFmembrane and probed with anti-caveolin-1 antibody. lane 1: NIH 3T3 lysate, no peptide added ; lane 2 : NIH3T3 lysate with biotinylated CBD-MA peptide; lane 3: NIH 3T3 incubated with biotinylated consensus CBD peptide; lane 3: positive control, NIH3T3 lysate, non-processed. Molecular weights are depicted (kDa).