Fig. 6. DRL is tyrosine phosphorylated in a SRC64B-dependent manner and DRL coexpression activates SRC64B. (A) DRL and SRC64B co-transfected cell lysates contain a predominant protein species with increased tyrosine phosphorylation (asterisk). Drosophila S2 cells were transfected with the indicated plasmids and WCEs analyzed by immunoblotting with an anti-phosphotyrosine mAb. (B) DRL is phosphorylated in a SRC64B-dependent manner. Lysates of S2 cells transiently transfected with the indicated constructs were immunoprecipitated with anti-DRL antiserum, complexes washed, disrupted by boiling and DRL reprecipitated with anti-HA antiserum and analyzed by anti-phosphotyrosine immunoblotting. V, vector alone. (C) Coexpression of DRL and SRC64B results in a WIF and cytoplasmic domain-dependent activation of SRC64B. S2 cells were transfected as follows: lane 1, SRC64B[WT] only; lane 2, DRL[WT] + SRC64B[WT]; lane 3, DRL[Cyto] + SRC64B[WT]; lane 4, DRL[WIF] + SRC64B[WT]; lane 5, DRL[WT] + SRC64B[KD]; lane 6, DRL[TBC] + SRC64B[WT]; and lane 7, DRL[PDZ] + SRC64B[WT]. WCEs were immunoblotted to detect active SRC64B (anti-PY434SRC64B), pan-SRC64B (anti-MYC) and DRL (anti-HA). All transfections contained SRC64B-MYC except lane 5. Quantitation of a similar experiment performed in triplicate is shown in Fig. S5 in the supplementary material. (D) DRL-associated SRC64B is, at least in part, catalytically active. Lysates from cells transfected as indicated were immunoprecipitated with anti-DRL and analyzed by anti-PY434SRC64B immunoblotting. Control anti-MYC (SRC64B) and anti-HA (DRL) blots are shown.