EGFP-DVL2 constructs (100 ng) and FLAG-Axin (300 ng) per six-well plate were cotransfected with Lipofectamine 2000 (Invitrogen), fixed, permeabilized, and stained 20 hours later with rabbit anti-GFP (green fluorescent protein) (G1544, Sigma) and mouse anti-FLAG antibody (F1804, Sigma), followed by Alexa Fluor 488–conjugated goat anti-rabbit (A11008, Invitrogen) and Alexa Fluor 594–conjugated goat anti-mouse secondary antibody (A11032, Invitrogen). COS-7 cells were then mounted in, and stained by, VECTASHIELD with 4′,6-diamidino-2-phenylindole (DAPI) and analyzed by confocal microscopy.

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