Figure 9

Oligomerization rate of wild type and mutant PPT1 molecules. (A) COS-1 cells were either single or double transfected with pCMV5-hPPT1 or with plasmids having the indicated mutations, together with GFP-vector (c) or with the corresponding GFP-PPT1 construct having the same mutation in the PPT molecule (m). The cells were lysed 48 h post-transfection. To verify the transfections, 10 μg of total protein from the lysates was analyzed by SDS-PAGE and immunoblotting using both anti-GFP antibody (not shown) and anti-PPT1 antibody (Transfection). The lysates were then subjected to immunoprecipitation using anti-GFP-agarose beads. Immunoprecipitates were analyzed by SDS-PAGE and immunoblotting using the anti-PPT1 antibody (IP). (B) The graph represents the dimerization degree of mutant proteins compared to wild type (calculated as a ratio between the immunoprecipitated GFP-PPT/PPT and the transfected GFP-PPT/PPT). An example of a single experiment's quantification is shown experiments.