Figure 2. KB02-SLF promotes the loss of nuclear FKBP12.
a, Structure of non-electrophilic control compound (C-KB02-SLF). b, Western blot of FLAG-FKBP12_NLS in HEK293T cells treated with KB02-SLF or C-KB02-SLF (2 µM, 8 or 24 h). Bar graph (bottom) represents quantification of the relative FKBP12 protein content, with DMSO-treated cells set to a value of 1. Data represent mean values ± SEM (n = 3 biologically independent experiments). Statistical significance was calculated with unpaired two-tailed Student’s t-tests comparing DMSO- to KB02-SLF-treated samples. **P < 0.01; ***P < 0.001. P values were 0.00036 and 0.0027. Full images of blots are shown in Supplementary Fig. 14. c, Structure of lenalidomide-SLF. d, Western blot of FLAG-FKBP12_NLS in HEK293T cells pre-treated with 1.5 µM of KB02-SLF or lenalidomide (Len)-SLF for 0.5 h, washed with PBS, and resuspended in fresh media for additional 7.5 or 23.5 h. Bar graph (bottom) represents quantification of the relative FKBP12 protein content, with DMSO-treated cells set to a value of 1. Data represent mean values ± SEM (n = 3 biologically independent experiments). Full images of blots are shown in Supplementary Fig. 14. e, Western blot of FLAG-FKBP12_NLS following treatment of HEK293T cells with KB02-PEG0-SLF, KB02-SLF, or KB02-PEG4-SLF (10) (0.5-2 µM, 8 h). The result is a representative of three experiments (n = 3 biologically independent experiments). Full images of blots are shown in Supplementary Fig. 14. f, Concentration-dependent degradation of FLAG-FKBP12_NLS by KB02-SLF in HEK293T cells (24 h treatment with indicated concentrations of KB02-SLF). The result is a representative of four experiments (n = 4 biologically independent experiments). Full images of blots are shown in Supplementary Fig. 14. g, Western blot of FLAG-FKBP12_NLS in HEK293T cells following treatment of HEK293T cells with KB02, KB02-PEG2 (11), SLF, or the combination of SLF and KB02 or KB02-PEG2 (1.5 µM, 8 h). The result is a representative of two experiments (n = 2 biologically independent experiments). Full images of blots are shown in Supplementary Fig. 14. h, Degradation of FLAG-FKBP12_NLS by KB02-SLF (2 µM, 8 h) is blocked by excess SLF (25 µM). The result is a representative of three independent experiments (n = 3 biologically independent experiments). Full images of blots are shown in Supplementary Fig. 14.