Sab4200007

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.

Cells were cultured and seeded at 37 °C under lower (2.5%O₂) and atmospheric (20%O₂) oxygen conditions. Once 70% confluent, cells were washed with PBS and harvested in RIPA buffer. Protein extracts were incubated with 4 μg of anti-DVL-1 antibody (D3570; Sigma) or anti-DVL-3 antibody (SAB4200007; Sigma) for 2 hrs/4 °C. Protein A dynabeads (Invitrogen) were added to the immune-complex and incubated for 2 hrs/4 °C. IP protocol was followed as mentioned above. Dry beads were shipped to Applied Biomics Inc. (Hayward, CA) for acetylation site identification by LC-MS/MS mass spectrometry.