Sensitive DVL2 Antibody

Results Summary

We treated HEK293T cells with 50% WNT3A-conditioned medium for 0, 0.5, 1, 2, 4, and 8 h. We then separated cytosolic and nuclear fractions by hypotonic swelling, needle shearing, and differential centrifugation. Cytosolic fractions (30 µg) and equal volumes of the corresponding nuclear fractions, representing approximately equal cell equivalents, were resolved on 4–12% Bis-Tris gels and transferred to nitrocellulose. Membranes were blocked in 5% milk/TBS-T and probed with the Dvl2 antibody at 1:1000 overnight at 4 °C, followed by HRP-conjugated donkey anti-rabbit IgG (1:10,000) and ECL (regular) detection. The antibody detected a strong band at approximately 95 kDa. This is above DVL2's predicted size of 79 kDa but typical for DVL proteins, which migrate anomalously in SDS-PAGE. DVL2 was found predominantly in the cytoplasmic fractions, with only a faint signal in the nuclear fractions. Note that we did not include a DVL2 knockout or knockdown control in this experiment. Because DVL1, DVL2, and DVL3 are closely related paralogs of similar molecular weight, a DVL2-null sample would have been the best way to confirm that the detected band is DVL2 and not a cross-reacting paralog. Readers who need definitive specificity should include such a control.