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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.
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