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. Author manuscript; available in PMC: 2017 Jan 24.
Published in final edited form as: Nat Protoc. 2016 Oct 27;11(12):2328–2356. doi: 10.1038/nprot.2016.114

Figure 5.

Figure 5

Assessment of N- versus C-terminal HaloTagging on T-REX functionality, exemplified by Keap1 LDE targeting. Results with Halo-Keap1 were previously reported56,57. (a) Live imaging analysis shows that Keap1-Halo promotes Nrf2 nuclear exclusion as with Halo-Keap1 (ref. 57). Inset shows quantification performed using ImageJ (NIH). ***, P < 0.001. Scale bars, 20 µm. (b) In-gel fluorescence analysis shows that targeted HNEylation of Keap1 in HEK-293 cells using the Keap1-Halo construct is equally as efficient as using Halo-Keap1 (refs. 56,57). L, MW ladder. (c) Ionization spectrum of Keap1 peptide modified by a representative cyclohexenone-derived LDE (CHE, Fig. 2 inset) as a result of T-REX on HEK-293 cells expressing C-terminal HaloTagged Keap1, subsequent enrichment of modified Keap1-Halo from T-REX-treated cells and LC-MS/MS analysis. The same Cys residue (C613) was modified in the corresponding experiment in which N-terminally HaloTagged-Keap1 was used56. Also see Supplementary Tables 1 and 2. Arrow points to the diagnostic m/z peak for C613 modification.