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. 2019 Jun 6;68(12):2214–2227. doi: 10.1136/gutjnl-2018-317872

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© Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/.

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Determination of two critical sites of activin receptor-like kinase 5 (ALK5) for glial cell line-derived neurotrophic factor (GDNF) binding. (A) The binding affinity of GDNF to ALK5 was determined using surface plasmon resonance (SPR). (B) The dissociation constant (KD) of GDNF and ALK5 was calculated by non-linear regression analysis. GDNF bound to ALK5 with a KD value of 85.47 nM. (C) The complex of ALK5 and GDNF was generated by in silico estimation using a rigid-body docking approach with PatchDock software (http://bioinfo3d.cs.tau.ac.il/PatchDock/). (D) Expression plasmid sequences of ALK5-wt and ALK5-mut were validated. (E) ALK5-wt and ALK5-mut amino acid sequences. (F) co-immunoprecipitation (co-IP) analysis of ALK5/GDNF interaction comparing wt and His39 and Asp76 mutated ALK5. LX2 cells were transfected with pcDNA3-ALK5 plasmids for 12 hours and subsequently treated with Ad-GDNF for an additional 48 hours. (G) Direct interaction of ALK5 with Ret after GDNF stimulation. After IP with an anti-ALK5 antibody, immunoblotting for Ret was performed. (H) Direct interaction of ALK5 with p-Smad2 after GDNF stimulation. After IP with an anti-ALK5 antibody, immunoblotting for p-Smad2 was performed.