Fig. 7. Mutation of putative phosphorylation sites in the long GalT I cytoplasmic domain affects its ability to act as a dominant-negative. Spreading of 3T3 cells transiently transfected with TS-GFP, TL-GFP or mutant constructs was measured in cells plated on laminin matrices as described in Materials and Methods. Spreading of TS-GFP-expressing cells was similar to mock and GFP transfectants, whereas cells expressing TL-GFP spread poorly, demonstrating the dominant-negative phenotype. Mutating S11 or T18 to aspartic acid (S11D or T18D) inhibited the dominant-negative phenotype, whereas substituting S11 and/or T18 with alanine (S11A or T18A and S11A,T18A) had no effect on the ability of the long cytoplasmic domain to produce a dominant-negative phenotype. Substituting F3 and F7 with glycine (F3G,F7G) also inhibited the dominant-negative phenotype, although this construct was expressed normally on the cell surface (Fig. 6). Data represents the mean ±s.e.m. from 3 individual determinations. P values were determined by Student's t test, and are shown for each construct. Using the Bonferroni method, statistical significance is calculated as P=0.05 divided by the number of mutants (in this case 6), therefore P<0.008 is statistically significant (asterisks).

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