Fig. 5: Detecting PPIs in C. elegans with SIMPL. | Nature Communications

Fig. 5: Detecting PPIs in C. elegans with SIMPL.

From: Split Intein-Mediated Protein Ligation for detecting protein-protein interactions and their inhibition

Fig. 5

a Scheme for creating and testing transgenic C. elegans lines for use with the SIMPL ELISA assay. Initially, 15 positive reference set (PRS) PPI pairs were successfully cloned and injected into C. elegans, along with 21 random (RRS) PPI pairs, in both the IN/IC and IN/CIC configurations (72 total lines injected). Potential transgenic lines were screened by western blot for expression of both bait and prey constructs, resulting in 10 total PRS lines and 13 total RRS lines (IC + CIC), representing 7 and 9 distinct PPI pairs, respectively. b Performance of the SIMPL ELISA assay in two biological replicates, with the fraction of PPI pairs that are positive at different cutoff values. Cutoffs were set as the ratio of spliced signal to total signal. A biological replicate was reported as the average value of two technical replicates. c Visualization of SIMPL ELISA results of the individually tested PPIs. The cutoff value used to determine whether a given PPI was positive or negative was 0.50 (arrow in b), which maximized the number of true-positive and true-negative interactions between both biological replicates. Pairs belonging to the RRS were never positive in both replicates compared to the PRS. Source data are available in the Source Data file.

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