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Fig. 4. Trio and Fra are tyrosine phosphorylated in S2 cells. (A,B) Pervanadate
treatment results in robust elevation of phosphotyrosine levels in both Trio
and Fra proteins. S2 cells transiently expressing Trio-Myc (A) or Fra-Myc (B)
were mock-treated with PBS (lane 1) or treated with pervanadate (lane 2) for
30 minutes. Target proteins were immunoprecipitated, and equivalent aliquots
of immune complexes were resolved by SDS-PAGE. Blots were probed with either
anti-phosphotyrosine (top gel) or anti-Myc (bottom gel). (C,D) Tyrosine
phosphorylation of both Trio (C, lane 2) and Fra (D, lanes 2 and 3) is
elevated in the presence of increased Abl levels (top gel). S2 cells were
co-transfected with 5 µg of pMET Trio-Myc (C) or pMET Fra-Myc (D) and 0, 2
or 5 µg of pMET Abl, as indicated. Twenty-four hours after induction,
target proteins were immunoprecipitated and resolved via SDS-PAGE. Blots were
probed with anti-phosphotyrosine (top gel), then stripped and re-probed with
anti-Myc to verify equivalent loading of samples (middle gel). Approximately
2% of total lysates used in each IP were resolved separately and elevated Abl
levels were verified with anti-Abl (bottom gel).
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