Fig. 5. SRC64B tyrosine kinase activity is required for the formation or stability of the DRL-SRC64B complex. (A) Herbimycin A treatment leads to significantly decreased amounts of SRC64B co-immunoprecipitating with DRL. Aliquots of Kc cells co-transfected with DRL-HA and SRC64B-MYC expression constructs were treated for 24 hours with increasing concentrations of herbimycin A or equivalent volumes of the DMSO carrier, WCEs prepared, DRL-SRC64B complexes immunoprecipitated with anti-DRL and then immunoblotted with anti-MYC (SRC64B) antibody (top panels). Lanes 1 and 5, untreated samples; lanes 2-4, herbimycin A-treated at 2.5, 5 and 10 µM final concentration, respectively; lanes 6-8, DMSO controls. Equal amounts of WCEs were evaluated for SRC64B-MYC expression (middle panels) and overall tyrosine phosphorylation levels (lower panels). (B) Wild-type (WT), but not kinase-dead (KD), SRC64B interacts with the intracellular domain of DRL (DRL-intra) in a mammalian two-hybrid assay. The indicated fusion protein constructs were transfected into SFK-deficient cells and luciferase activity was measured 48 hours post-transfection and plotted, normalized to an internal control. Immunoblotting for DRL and SRC64B species (inset) indicates equivalent expression of the test plasmids. An irrelevant lane between lanes 5 and 6 was removed in preparing the panel.