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. 2011 Oct 26;6(10):e26636. doi: 10.1371/journal.pone.0026636

Figure 10. Porcn overexpression increases the association of spGFP∶Wnt1 (209–239), but not spGFP∶Wnt1 (35–108) with lipid rafts.

Figure 10

HEK293T cells were transiently transfected with selected spGFP∶Wnt1 constructs along with empty vector (Cntrl) or Porcn. Cell lysates were separated by ultracentrifugation through an Optiprep density gradient. Fractions from the gradient were collected from top (1) to bottom (10). Western blots for Wnt1, Flotillin, and PCNA are shown in Panel A. Western blot data was then quantitated using NIH Image J. The graphical representations are shown in Panel B, where each bar corresponds to the percent of total protein in a particular fraction. The percentage of protein directed to fraction 3 (the primary lipid raft containing fraction) in a Porcn-dependent manner was calculated by subtracting the percentage of protein in Cntrl transfected cells from that in Porcn transfected cells (Panel C). The data in Panels B and C represent the average values obtained from 2–3 independent experiments. The co-transfection of cells with Porcn and SpGFP∶Wnt1 (35–108) had no effect on the levels of this fusion protein in lipid rafts. Co-transfection of cells with Porcn and spGFP∶Wnt1(209–239) or spGFP∶Wnt1(209–239) S224A did increase the relative proportion of fusion protein associated with lipid rafts.