Supplementary Figure 1: The design and in vitro validation of INT-Ub. | Nature Methods

Supplementary Figure 1: The design and in vitro validation of INT-Ub.

From: Internally tagged ubiquitin: a tool to identify linear polyubiquitin-modified proteins by mass spectrometry

Supplementary Figure 1

(A) Ub chain building potential of various Ub mutants (top). Ub molecule with an insertion point of STREP-tag II (black) and the following color-coded residues: K (red), M (orange), G76 (blue) and I44 (yellow). This Figure was generated with PyMOL software by modifying a 1UBQ.pdb file (bottom). (B) Coomassie staining of recombinant Ub variants used for CD measurements in (C) Far-UV CD spectra of wild-type Ub (blue), Ub.7KR (red), INT-Ub (green) and INT-Ub.7KR (violet). (D) and (E) A direct comparison of in vitro ubiquitination reaction for untagged Ub, INT-Ub and INT-Ub.ΔGG. (F) Pull-down assay between INT-Ub and UBDs that can recognise single Ub moieties. (G) A direct comparison of diUb and INT-diUb ability to bind linear polyUb-specific UBD UBAN. (H) A direct comparison of USP2-cc DUB assay with diUb and INT-diUb. (I) A direct comparison of OTULIN DUB assay with diUb and INT-diUb. Blot and gel images for Supplementary Figure 1b,f-i were cropped to improve the conciseness of the presentation (full blots and gel images are available in Supplementary Figure 12).

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