Fig. 7. (A) The YFP-EBP50 mutant lacking the ERM-binding site still associates with TRPC4 in HEK293 cells. Extracts of HEK293 cells expressing myc-TRPC4 together with YFP-EBP50 or YFP-ERM were incubated with an anti-YFP antibody and precipitated using Protein-G-coated agarose beads. Bound proteins were resolved on SDS-PAGE, transferred to PVDF and probed with an anti-myc antibody (lower panel). The upper and middle panels show the levels of expression of myc-TRPC4 and the YFP-tagged proteins, respectively, in the transfected cells. (Upper panel) 15% input; (Lower panel) 40% precipitate. IB, immunoblot; IP, immunoprecipitation. (B) Deletion of the ERM-binding site decreases the membrane-associated fraction of YFP-EBP50. Western blots of total extracts (T.E.) and crude membrane fractions (Mb.) prepared from HEK293 cells expressing myc-TRPC4 or myc-TRL together with either YFP-EBP50 or YFP-ERM. The amount of loaded proteins is indicated (µg). (Upper panels) Blots probed with an anti-myc antibody to detect myc-TRPC4 and myc-TRL; (Lower panels) blots probed with an anti-YFP antibody to detect YFP-EBP50 and YFP-ERM. Molecular weights as revealed by prestained protein markers are indicated to the right (kDa). Results shown are representative of three independent experiments.