SW480 cells were cultured at 37° C at normal atmospheric levels of CO2 in L15-media (Cellgro) supplemented with 10% FBS and 1x penicillin–streptomycin. Drosophila APC2 or Axin protein constructs were transfected into SW480s using Lipofectamine 2000 (Invitrogen) as recommended by the manufacturer. Cells were imaged 24 hours later. Full length Drosophila APC2 and Axin were cloned with either a GFP, RFP, or Flag tag as in [17 (link)].
To verify that Axin:GFP polymerization is not simply a result of di- or oligomerization of the GFP protein, we created a monomeric GFP (mGFP) by changing Alanine 206 to Leucine [46 (link)]. We created the A206K amino acid change in our base plasmid (pCMV-Axin:GFP; [17 (link)]) using QuikChange (Agilent Technologies) following manufacturer’s recommendations. The full plasmid was sequenced to verify the amino acid change in GFP and to ensure no other detrimental mutations were induced in the plasmid.
To verify that Axin:GFP polymerization is not simply a result of di- or oligomerization of the GFP protein, we created a monomeric GFP (mGFP) by changing Alanine 206 to Leucine [46 (link)]. We created the A206K amino acid change in our base plasmid (pCMV-Axin:GFP; [17 (link)]) using QuikChange (Agilent Technologies) following manufacturer’s recommendations. The full plasmid was sequenced to verify the amino acid change in GFP and to ensure no other detrimental mutations were induced in the plasmid.
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