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Extended Data Fig. 7 | BIK1 in vivo ubiquitination sites identif ied by mass
spectrometry. a, Ubiquitinated BIK1–GFP in planta was immunoprecipitated
for LC–MS/MS analysis. BIK1–GFP and FLAG–UBQ were co-expressed in
wild-type protoplasts (about 4 × 10^6 cells) and then treated with 200 nM
f lg22 for 30 min. Ubiquitinated BIK1 was immunoprecipitated with
GFP-trap-agarose, separated by SDS–PAGE, digested with trypsin and
subjected to LC–MS/MS analysis. Portions of cell lysates were examined for
BIK1–GFP expression (left), and immunoprecipitates were analysed 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 analysed by MS. b, BIK1 is ubiquitinated in vivo. Ubiquitinated lysines
containing a diglycine remnant identified by LC–MS/MS analysis are marked in
red with amino acid positions. c–h, MS/MS spectra of peptides containing
ubiquitinated lysines of BIK1 are shown. c, K31; d, K41; e, K95; f, K337; g, K358;
h, K366. MS spectra are outputs from the SEQUEST program. MS analysis was
performed once.