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. 2022 Oct 31;11:e80895. doi: 10.7554/eLife.80895

Figure 6. Diversity in DARPin-hFc clone-specific interactomes reveals putative isoform-specific gephyrin interactors.

(A) Canonical and non-canonical (metabolic, mRNA binding, and ribosomal ontology) gephyrin interactors show binder-specific abundance profiles. Only significantly regulated interactors are shown. (B) DARPin-hFc clones 27B3 and 27G2 recognize both full-length gephyrin and the GC-domain while clone 27F3 recognizes only full-length gephyrin suggesting different binding epitopes. (C) DARPin-hFc 27F3 only recognizes the principal P1 (synaptic) isoform of gephyrin while clones 27B3 and G2 additionally recognize non-neuronal isoforms containing the C3 cassette. (D) DARPin-hFc 27F3-determined gephyrin interactome enriched over-representation analysis of biological processes. (E) DARPin-hFc 27B3 and 27G2-determined gephyrin interactome enriched over-representation analysis of biological processes. Statistics: (A) two-way ANOVA with multiple-comparisons correction comparisons all groups, three replicates per group.

Figure 6—source data 1. Values used to generate heat maps in Figure 6A.

Figure 6.

Figure 6—figure supplement 1. DARPin-specific gephyrin interactor abundance.

Figure 6—figure supplement 1.

Common gephyrin interactors identified by all DARPin-hFc-based interactomes showing proteins with significantly different abundances relative to gephyrin, organized by hierarchical clustering. Only significantly regulated interactors are shown. Statistics: two-way ANOVA with multiple-comparisons correction comparisons all groups, three replicates per group.
Figure 6—figure supplement 1—source data 1. Values and statistical test results indicating differentially abundant gephyrin interactors between binding experiments.
Figure 6—figure supplement 2. Identification of gephyrin-binding preferences of anti-gephyrin DARPins using an in-cell HEK293T fluorescence assay.

Figure 6—figure supplement 2.

(A) Representative images of eGFP-gephyrin expressed in HEK cells that were fixed and probed using DARPin-FLAG clones or commercial antibody clone 3B11. Shown is eGFP and FLAG signal provided by the control (E3_5) and gephyrin-binding DARPin-FLAG clones (e.g., 27B3). The relative signal between eGFP and FLAG for a given cell is plotted, and the slope compared between clones to assess relative binding. (B) Quantification of binder labeling of eGFP-tagged gephyrin WT versus S268A/S270A and S268E/S270E phospho-mutants overexpressed in HEK293T cells. (C) Quantification of binding to overexpressed full-length (P1 variant) gephyrin or GC or E domains only. (D) Quantification of binding to eGFP-tagged gephyrin P1 isoform or isoforms including the C3 or C4a cassettes. Statistics: one-way ANOVA, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Data points represent the slope calculated from at least 25 cells in three independent experiments. All panels: mean and SD are presented.
Figure 6—figure supplement 2—source data 1. Values and statistical analysis performed to generate graphs in Figure 6—figure supplement 2B–D.
Figure 6—figure supplement 3. Non-neuronal interactor ontology.

Figure 6—figure supplement 3.

Heat map of relative abundance of proteins of ‘myelin sheath’ or ‘glial projection’ ontology between different DARPin-detected interactomes; gray squares indicate that the binder was not detected as a gephyrin interactor using a given DARPin.
Figure 6—figure supplement 3—source data 1. Values used to generate heat maps in Figure 6—figure supplement 3.
Figure 6—figure supplement 4. Relative C3 cassette recovery.

Figure 6—figure supplement 4.

Relative recovery of C3-containing gephyrin peptide fragments relative to total gephyrin from liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. N = individual experimental replicates. Statistics: one-way ANOVA, *p<0.05, **p<0.01, mean and SD are presented.
Figure 6—figure supplement 4—source data 1. Values used to generate graphs in Figure 6—figure supplement 4.