Fig 5. Comparison of Git1 phosphorylation at Tyr-554 and its binding to paxillin between wild-type and Ptprz-deficient mice.

A, Western blotting of cerebral synaptosome extracts prepared from the brains of wild-type (+/+) and Ptprz-deficient (−/−) mice. The overall tyrosine phosphorylation pattern and protein expression were analyzed with an anti-phosphotyrosine PY20 antibody, and rabbit anti-Git1 and anti-paxillin antibodies, and rabbit anti-Ptprz-S, respectively. The Ptprz-B isoform was previously shown to be detectable after the chondroitinase-ABC treatment [16] because Ptprz proteins are expressed as chondroitin-sulfate proteoglycans in the brain [15]. B, Tyr-554 phosphorylation of Git1. The synaptosome extracts were immunoprecipitated with rabbit anti-GIT1/Cat-1 antisera-coated beads, and the binding proteins were analyzed by Western blotting with rabbit anti-pY554-Git1 and mouse anti-Git1 antibodies. The Tyr-554 phosphorylation level was determined by densitometric analyses. Data are the mean ± S.E. (error bars; n = 5). *, P < 0.05 significantly different from the wild-type by the Student’s t-test. C, Git1 binding to paxillin. The immunoprecipitated proteins with mouse anti-paxillin were analyzed by Western blotting with rabbit anti-Git1 and rabbit anti-paxillin antibodies. The amount of Git1 relative to that of paxillin was determined by densitometric analyses. Data are the mean ± S.E. (error bars; n = 6). **, P < 0.01 significantly different from the wild-type by the Student’s t-test.