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. Author manuscript; available in PMC: 2020 Oct 22.
Published in final edited form as: Nature. 2020 Apr 22;581(7807):199–203. doi: 10.1038/s41586-020-2210-3

Extended Data Figure 7. BIK1 in vivo ubiquitination sites identified by mass spectrometry.

Extended Data Figure 7.

a. Ubiquitinated BIK1-GFP in planta was immunoprecipitated for LC-MS/MS analysis. BIK1-GFP and FLAG-UBQ were co-expressed in WT protoplasts (~4 × 106 cells) followed by 200 nM flg22 treatment for 30 min. Ubiquitinated BIK1 was immunoprecipitated by GFP-trap-agarose, separated by SDS-PAGE, digested by trypsin and subjected to LC-MS/MS analysis. Portions of cell lysates were examined for BIK1-GFP expression (left), and immunoprecipitates were analyzed by SDS-PAGE following silver staining (middle, right for longer exposure of the same gel) and SDS-PAGE following CBB staining (right). The highlighted area was cut and analyzed by MS. b. BIK1 is ubiquitinated in vivo. Ubiquitinated lysine containing a diglycine remnant by LC-MS/MS analysis are marked as red with amino acid positions. c to h. MS/MS spectrums of peptides containing ubiquitinated lysine of BIK1 are shown. c. K31; d. K41; e. K95; f. K337; g. K358; h. K366. MS spectrums are outputs from the SEQUEST program. MS analysis was performed once.