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. Author manuscript; available in PMC: 2016 Apr 1.
Published in final edited form as: Int J Biochem Cell Biol. 2015 Jan 23;61:29–34. doi: 10.1016/j.biocel.2015.01.012

Fig. 1. CAR has a high affinity interaction with MAGI-1 PDZ3.

Fig. 1

A) Yeast Y187 cells were transformed with pairs of pGBKT7-derived bait (CAR C-terminus) and pGADT7-derived prey vectors (PDZ domains), and plated on selective media containing X-α-gal. Blue colony indicates an interaction between CAR and PDZ3. B) In vitro pull down assay. Individual 35S-methionine-labeled MAGI-1 PDZ domains generated by in vitro translation were added to immunoprecipitated FLAG-tagged CAR from transfected COS-7 cells (CAR lysate). Western blot for immunoprecipitated CAR (FLAG-CAR), and autoradiography for PDZ domain binding (N, no PDZ domain). In vitro translation of all PDZ domains was confirmed by autoradiography. Emission spectra, linear plot of binding curve and average change in maximal fluorescence intensity at 570 nm (F0-F) of Cy3-PDZ domains and Cy5 CAR C-termini upon excitation of Cy3 at 550nm with increasing concentration of Cy5 CAR C-termini (0-500nM). C) Cy3-PDZ1 and Cy5-CAR (no interaction). D) Cy3-PDZ3 and Cy5-CAR. All emission spectrum were corrected for background signals. NA = no interaction. Representative data shown from 3 replicate experiments.