Fig. 1.

Quantitative and comparative indexing of locale-specific native first responders. Also see SI Appendix, Figs. S1–S13. (A) Localis-rex setup for indicated subcellular locales. (Left) Mitochondrial outer membrane (MOM) vs. nuclear-targeted spatiotemporally controlled as well as dosage-controlled release of electrophilic signals, exemplified by alkyne-functionalized hydroxynonenal (HNE) (red dot). MOMLS, MOM-localization sequence. NLS, nuclear-localization sequence. Ht-PreHNE, Halo-targetable photocaged HNE(alkyne). (Right, Inset) A representative Localis-rex strategy (SI Appendix, Fig. S1A), coupled with the established target- and signal-specific pathway-interrogation method, T-REX (10, 23) (SI Appendix, Fig. S1B). (B) Validation of candidate first responders by T-REX (10, 23) in HEK293T cells expressing the respective Halo-TeV-Flag2-POI (POI, protein of interest; “TeV” designates a linker housing a TeV-protease-cleavable site, here and elsewhere) by Click-biotin pulldown assay followed by Western blot analysis using indicated antibodies. Cell lysates were subjected to Click coupling (with biotin-azide), protein precipitation, resolubilization, streptavidin enrichment, washing, and elution, followed by Western blot analysis. (C) Determination of fractional ligand occupancy on CDK9 following T-REX (10, 23, 49) in HEK293T cells expressing Halo-TeV-Flag2-CDK9 (against controls: light alone, Ht-PreHNE-probe alone; see SI Appendix, Fig. S1 B, Inset). (Left) Representative data from Click-biotin pulldown assay (49) followed by Western blot analysis using indicated antibodies. (Right, Inset) Quantitation. Hydroxynonenal occupancy was calculated as the ratio of [“Flag2-CDK9” precipitated post–TeV-mediated separation of Halo and CDK9 in T-REX–treated samples]:[“Halo-TeV-Flag2-CDK9” precipitated from light-protected and non–TeV-treated samples], which is equivalent to (hydroxynonenylated-CDK/total CDK9). (n = 3 biological replicates; error bars, SD; two-tailed t test was applied.)