Fig. 6: SIMPL for enzymatic/PPI inhibitor identification.

a Time schedule for studying enzymatic/PPI inhibitors with SIMPL. To avoid splicing before inhibition can occur, an inhibitor has to be administered before protein expression. As the expression is under the control of Tet-on promoter, tetracycline is added to the cells alongside the inhibitor 6 h before assay. b Studying an EGFR kinase inhibitor with SIMPL. EGFR kinase inhibitor AG1478 at different indicated doses was incubated with the cells expressing EGFR-IN and IC-SHC1. The spliced EGFR-SHC1 band observed by western blot diminished with increasing AG1478 concentration. The blot is representative of three independent experiments. c–e BAX/BCL2 interaction was assayed in different formats as indicated by western analysis. In the case of NIN-BAX/IC-BCL2, immunoprecipitation was performed to resolve the spliced protein from its parental protein since they have similar mobility upon electrophoresis (e). Each blot is representative of three independent experiments. f Heatmap of SIMPL ELISA readings of BAX/BCL2 interaction in different formats. Gray color: not tested. LSM3 and LSM2 were used as negative controls for bait and prey respectively. g Investigation of the BCL2/BAX PPI inhibitor venetoclax with SIMPL. Cells expressing NIN-BAX and IC-BCL2 (or BCL2L1 as control) were treated with venetoclax (or osmeritinib as a negative control) at different concentrations as indicated. The cells were then subjected to ELISA analysis. SIMPL signals were normalized to bait expression. The experiment was performed in triplicates and each replicate is presented as a single dot. Source data are available in the Source Data file.