D3570

8 × 10⁵ cells were seeded onto coverslips (12mm) in a 60mm tissue culture dish. The cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature, followed by a wash with PBS for 5 minutes, a quench step with 50mM ammonium chloride (NH₄Cl) in PBS for 5 minutes with an additional 5 minutes PBS wash. The coverslips were blocked with 5% Bovine serum albumin (BSA) in PBS (blocking buffer) for 30 minutes, followed by an hour incubation with the following primary antibodies in 5% BSA in PBS: DVL-1 (D3570; Sigma) and DVL-3 (SAB4200007; Sigma). The samples were rinsed 3 times with PBS and then incubated with secondary antibodies purchased from ThermoFisher scientific (Alexa flour 568 #A11036, Alexa fluor 647 #A21235 and Alexa fluor phalloidin 488 #A12379 from Thermo Scientific) for 1 hour at room temperature. The samples were rinsed several times in PBS for 5 minutes each and then mounted with prolong gold antifade mounting solution with DAPI (P36941, Thermo Scientific), then cured overnight at room temperature and stored at −20°C until imaged. The samples were imaged using a laser scanning confocal microscope Nikon T-1E with a 60x objective and NIS software.

For endogenous β-catenin, DVL1, DVL2 and DVL3 level detection, SH-SY5Y cells were plated in 24-well tissue plates and transfected with 400ng FLAG-ALFY constructs (wild type, mutant, Δ-FYVE and mock). After an ON incubation with the plasmid mix, cell's medium was replaced with Wnt3a conditioned medium for 8 hours. Total protein lysates were collected for further analysis. Primary antibodies used for this experiment: Mouse anti-FLAG (1:3000-Sigma-Aldrich), Rabbit anti-DVL1 (1:1500-D3570, Sigma-Aldrich), Rabbit anti-DVL2 (1:10000-D1321, Sigma-Aldrich), Rabbit anti-DVL3 (1:6000-SAB4200008, Sigma-Aldrich), Mouse anti-DVL3 (1:500-sc-377289, Santa Cruz), Rabbit anti-β-catenin (1:7000-ab32572, abcam), HRP (Goat) anti-Actin (1:6000-sc-1616, Santa Cruz). Secondary antibodies: HRP (Goat) anti-mouse (1:10000—sc-2005, Santa Cruz), HRP (Donkey) anti-rabbit (1:30000 –ab97085, abcam).