Specific Dephosphorylation at Tyr-554 of Git1 by Ptprz Promotes Its Association with Paxillin and Hic-5
Fig 7
Git1 Tyr-554 mutants failed to restore impaired cell motility by Git1 knockdown in a random cell motility assay.
A, Western blotting of Git1 expression in parental A7r5 cells, Git1-knockdown (Git1-KD) cells, and Git1-KD cells exogenously expressing the mCherry-fused Git1 mutants (WT, Y554F, Y554D, and Y9F-Y554). Both the endogenous Git1 and mCherry-fused Git1 mutant proteins were detected with anti-Git1. The protein amounts applied were verified by CBB staining. B, Wind-Rose plots of individual cell trajectories on fibronectin-coated dishes. Live-cell images were taken every 5 min for 3 h per experiment. Before beginning the recording, the expression of mCherry-fused proteins was verified in the rescue experiments by a short exposure to UV to avoid possible cell damage. Each wind-rose plot showed tracks from a total of 23 cells per group, with the initial position of each track being superimposed at a common origin for clarity. Scale bars, 100 μm. The lower graph shows the length along the trajectory. Data are the mean ± S.E. (error bars; n = 23). **, P < 0.01 significantly different from parental cells; #, P < 0.05 significantly different from Git1-KD cells by ANOVA and Scheffé’s post hoc tests.