Fig. 1. TRPC4 associates with both PDZ domains of EBP50 through its C-terminal TRL motif. Triton extracts of HEK293 cells expressing either myc-TRPC4 or myc-TRL were incubated for 3 hours at 4°C with glutathione-sepharose beads loaded with either GST, GST-PDZ1 or GST-PDZ2. After extensive washing, bound proteins were analyzed by SDS-PAGE, transferred to PVDF and probed with an anti-myc antibody (right panel). The middle panel demonstrates that myc-TRPC4 and myc-TRL were expressed at comparable levels in the transfected cells. Note that control cells (lane 4) transfected with the empty vector failed to react with the anti-myc antibody, implying that the immunoreactivity is specific for the myc epitope. The left panel shows the amount of GST or GST-fusion proteins used, as revealed by Coomassie Blue staining. Lanes 4-6 contain 10% of the extract used for the binding assay and lanes 7-12 contain 40% of the eluate. Results shown are representative of three independent experiments.

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