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. Author manuscript; available in PMC: 2019 Dec 17.
Published in final edited form as: Nat Chem Biol. 2019 Jun 17;15(7):737–746. doi: 10.1038/s41589-019-0279-5

Figure 5. Evaluation of DCAF16 cysteines involved in KB02-SLF-induced degradation of FKBP12.

Figure 5.

a, MS/MS spectrum of KB02-PEG0-SLF-modified, triply charged DCAF16 peptide (amino acids 168–184; see Supplementary Fig. 9b). The m/z 1235.4 ion represents a doubly charged DCAF16 peptide (168–184) with a cleaved probe adduct that occurs in MS/MS. The b- and y-ions are shown along with the peptide sequence. The experiment is repeated twice independently with similar results. b, Western blot of FLAG-FKBP12_NLS in DCAF16−/− cells (clone 4) expressing WT or C58S, C100S, C103S, C119S, C173S, C177S, C178S, or C179S mutants of HA-DCAF16 (or empty pRK5 vector control) following treatment with KB02-SLF (1.5 µM, 8 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 < 0.05; **P < 0.01; ***P < 0.001. P values were 0.00098, 0.0024, 0.0011, 0.00057, 0.019 and 0.00028. Full images of blots are shown in Supplementary Fig. 14.