Extended Data Fig. 4: Validation of GR as import shuttle for FKBP12.
From: Targeted protein relocalization via protein transport coupling
a, TRAM engaging GR and FKBP12. b, c Unlinked warhead controls for GR-based TRAMs. 8 binds to GR and 9 binds to FKBP12. d, Nuclear hormone receptor (ERα, GR) hijacking for import of target proteins. e, Nuclear localization of mCherry-FKBP12 after treatment with 6 or unlinked warheads 8 and 9 for 3 h. f, Nuclear localization of GR after treatment with 6 or unlinked warheads for 3 h. g, Genetic construct for stable incorporation. h, Representative live-cell images of GR-dependent FKBP12 import upon treatment with 6 or unlinked warheads 8 and 9 for 3 h. i, FKBP12 nuclear import dose response derived from the analysis pipeline after a 3-hour treatment with 6 engaging GR. j, Nuclear mCherry localization upon treatment with 6 in competition with either 1x or 10x of 8 and 9 for 3 h. k, Nuclear localization of GR upon treatment with 6 in competition with either 1x or 10x of 8 or 9 for 3 h. l, Representative live-cell images of the unlinked warhead competition assay for GR-dependent FKBP12 import after a 3-hour treatment. Images in h, l, are representative of three biological replicates. Data in e, f, i, j, k are mean ± s.d. of three independent experiments. Scale bars are 20 µm. P values in e, f, i, j, k were determined by one-way ANOVA with Dunnett’s post hoc test comparing each condition to the DMSO control. Hoechst stains the nucleus.
