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. 2020 Jan 21;117(5):2693–2703. doi: 10.1073/pnas.1911553117

Fig. 7.

Fig. 7.

A quantitative interaction screen identifies SERK proteins as putative coreceptors for GSO1/SGN3. (A) Schematic overview of the biochemical screen for a GSO1/SGN3 coreceptor. Streptavidin (in gray) was immobilized by using amine coupling as described in Fig. 1A. The GSO1/SGN3 ectodomain was captured by streptavidin on the GCI chip surface (in blue), the CIF peptide is provided in access in the running buffer (in black), and different recombinantly purified coreceptor candidates are assayed for binding (in orange). (B) Coomassie-stained SDS/PAGE of the receptor and coreceptor candidates used in the screen. Each lane depicts 1 µg of the LRR ectodomain of each indicated candidate. Shown are isolated monomeric peak fractions from size-exclusion chromatography experiments. (C) GCI assays of SERK1 LRR-RK ectodomain versus the SGN3 wild-type and mutant ectodomains in the presence of CIF2 variant peptides. The remaining candidates are shown in SI Appendix, Fig. S10. Coreceptor candidates were supplied at a flow rate of 25 µL min−1. Sensorgrams are shown with raw data in red and their respective fits in black. A 1-to-1 binding model was used for analysis. Table summaries of kinetic parameters are shown. (D) Complex formation of SERK1 and SGN3 ectodomains. (D, Left) Analytical size-exclusion chromatography traces of the SGN3 ectodomain in the absence (blue line) or presence of CIF2WT peptides (red dotted line) or CIF2I81D antagonistic peptides (black dotted line). An SDS/PAGE analysis of the corresponding fractions is shown alongside. The theoretical molecular weight is 94.1 kDa for SGN3 (residues 19 to 870) and 21.5 kDa for SERK1 (residues 24 to 213), respectively. (E) Induced barrier defect in inducible SERK3 dominant negative lines. Quantification of barrier permeability was done using the PI assay (n ≥ 8 for each condition). Shown are box plots spanning the first to third quartiles, with the bold line representing the median and circles indicating the raw data. Whiskers indicate maximum and minimum values, except outliers (b statistically significant difference from a, with P < 0.05, one-way ANOVA and Tukey test).