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Figure 3


Fig. 3. The conserved histidine residue in the predicted porcupine active site is required for the lipid-modification of Wnt3a. 293T cells were transfected with chick (c) Wnt3a in the presence of either GFP (control), mouse (m) PorcD, mPorcD(H341D) or mPorcD(H341Q). Cells were lysed 24 hours post-transfection and extracted with TX-114 before analysis by SDS-PAGE and western blot. The blots were probed with Wnt3a and tubulin (control) antibodies. Monomeric Wnt3a proteins migrate at ~40 kDa, whereas tubulin migrates at 50 kDa. In the presence of GFP alone, Wnt3a is in both aqueous (A) and detergent (T) phases. Whereas the presence of ectopic porcupine promotes the partitioning of Wnt3a into the hydrophobic phase, ectopic mPorcD(H341D) and mPorcD(H341Q) failed to show a similar effect.





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