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).