Fig. 1: Development of the SIMPL assay.

a The design of SIMPL for PPI detection. b Schematic representation of SIMPL bait and prey constructs and the resplitting of the GP41-1 intein. c Examination of SIMPL system with GP41-1 split intein with different splitting sites. DNA constructs coding for FRB-IN and IC-FKBP1A with inteins split at the sites were expressed in HEK 293 cells. After incubation with rapamycin (100 nM) for 2 h, the cells were lysed and the lysates subjected to western blot analysis with α-V5 and α-FLAG antibodies. Both IN and IC constructs with C25 splitting exhibited the best performance and were adopted as the standard sensor intein for the SIMPL platform. The blot is representative of four independent experiments. d Dose response of rapamycin-induced FRB/FKBP1A interaction examined with SIMPL. HEK 293 cells expressing FRB-IN and IC-FKBP1A were treated with the indicated doses of rapamycin for 2 h followed by western blot analysis. The densities of spliced bands (FRB-FKBP1A) were quantified with ImageJ and are presented as bar graphs above the blots. The blot is representative of three independent experiments. e Time course of rapamycin-induced FRB/FKBP interaction. HEK 293 cells expressing FRB-IN and IC-FKBP1A were treated with rapamycin (100 nM) for different periods of time as indicated followed by western blot analysis. The densities of spliced bands (FRB-FKBP1A) were quantified with ImageJ and presented as bar graphs above the blots. The blot is representative of five independent experiments. f Stable cells derived from HEK 293 T-Rex FlpIn with FRB-IN and IC-FKBP1A inserted into the FRT site were treated with the indicated different concentrations of tetracycline for 16 h, followed by treatment with rapamycin (100 nM) for 2 h and then analysis by western blot. HEK 293 cells transiently transfected with FRB-IN and IC-FKBP1A were used as a control (right two lanes). The blot is representative of three independent experiments. Source data are available in the Source Data file.