<p>(A) Immunofluorescence of human SW480 cells transfected with a plasmid encoding GFP (3 kb; Miraprepped using 1x volume of ethanol+50 μg/ml RNase) and stained for β-catenin via antibody. SW480 cells have high levels of the Wnt transcriptional co-activator β-catenin due to a mutation in one of its key negative regulators, APC. (A’) GFP is uniformly distributed throughout transfected cells. (A”) Expression of GFP does not alter β-catenin levels—arrows compare a transfected and an untransfected cell. (B) Immunofluorescence of SW480 cells transfected with a plasmid encoding GFP-tagged <i>Drosophila</i> APC2 (8 kb; Miraprep using 1x volume of ethanol+50 μg/ml RNase). (B’) APC2 is uniformly distributed in the cytoplasm. (B”) Fly APC2 is able to reduce β-catenin levels, thus compensating for the mutation of the endogenous human APC in the SW480 cells. (C) Transfection efficiency into SW480 cells is similar for Miraprepped samples (using 1x volume of ethanol+50 μg/ml RNase) and those transfected with DNA prepared via the standard Qiagen Maxiprep procedure. 2 μg of plasmids encoding GFP (3 kb) or GFP-tagged Drosophila APC2 (8 kb) were transfected using Lipofectamine 2000. 100 cells were counted in each of three independent experiments. (D) Immunoblot analysis of transfection efficiency. 3 conditions were tested: DNAs prepared by Miniprep (GeneJET), Miraprep (using 1x volume ethanol), and Miraprep (using 1x vol+50 μg/ml RNase). All led to roughly equal levels of protein expression. Lipofectamine 2000 and 2 μg of plasmid DNA were used. Cells were directly lysed in SDS-loading buffer. aPKCγ was used as the loading control.</p
