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. Author manuscript; available in PMC: 2020 Apr 23.
Published in final edited form as: Curr Biol. 2019 Sep 26;29(19):3256–3265.e5. doi: 10.1016/j.cub.2019.08.032

Figure 4. LRE-like GPI-APs Function as Co-receptors in RALF-CrRLK1L Complexes.

Figure 4.

(A and B) In vitro binding assays with E. coli-expressed His-tagged LLG2 (A) and LLG3 (B) proteins. Both RALF4/19 were biotinylated, and pull-down assays were performed with streptavidin magnetic particles. Western blots were performed using an α-His antibody.

(C and D) Pull-down assays of His-tagged LLG3 and FLAG-tagged ectodomains of BUPS1/2 (C) and ANX1/2 (D) with or without the addition of RALF4/19/34 peptides (each 100 nM) as indicated. Pull-down assays were performed with Ni Sepharose, and western blots were probed with antibodies against FLAG and His (α-FLAG or α-His), respectively.

(E and F) Interactions between His-tagged LLG2/3 with ectoFER-MBP by pull-down assays in the presence or absence of RALF23 peptide (100 nM). (E) Pull-down assay using LLG1-His and FLAG-tagged BUPS1/ANX ectodomains with/without 100 nM RALF4 (F). LLG1/2/3-His and ectoFER-MBP were expressed in E. coli, and ectodomains of BUPS1/ANX1 were generated after transient expression in tobacco leaves. Pull-downs were performed with Ni Sepharose, and western blots were probed with antibodies against MBP, FLAG, and His (α-MBP, α-FLAG, or α-His), respectively.

All experiments were repeated at least three times with similar results.