Fig 1: ß-cat phosphorylation at Y654 is enhanced by basic amino acid replacement at T653. (a) Four ß-cat constructs were transfected and expressed equally in HEK 293 T cells. (b) Co-transfection of Src/CA promotes ß-cat Y654 phosphorylation. Site-specific antibody does not recognize mutants. (c) Equal amount of lysates (1 mg) were immunoprecipitated with a FLAG antibody. Pan-phosphotyrosine antibody displays different binding affinity to WT and mutants.(d) ß-cats were immunoprecipitated with pY and blotted for FLAG to quantify their Y654 phosphorylation. (e) pY654 is quantified by first taking the ratio of FLAG signal in pY-pulldown and the lysate input to account for loading/expression differences. Then, the corrected intensities of mutants were compared to WT. In biological triplicate experiments, both basic ß-cat T653 mutants are more phosphorylated than WT. Error bars: one standard deviation. *: p < 0.05 in t-test. (f) ß-cat pY654 is a substrate for SHP1. (g) Inhibiting PTPs with pervanadate (100 µM) eliminates the difference in Y654 phosphorylation between WT and mutants.