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. 2013 Apr 5;288(21):15181–15193. doi: 10.1074/jbc.M112.446534

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© 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 3. — Amot130 is ubiquitinated at lysine 481 by AIP4. A, the y and b ion MS/MS spectra of the VSEAYENLVK*SSSKR trypsinized peptide. Lysine (y6 + 1) is shifted from 692 to 805 m/z; a signature for ubiquitinated residues (Lys + Gly + Gly (K-G-G)). B, ubiquitination of YFP-tagged Amot130 and Amot130 mutants P-Y2,3F (Y242F and Y287F), K481R, and P-Y2,3F/K481R following co-expression with FLAG-tagged AIP4 was examined following immunoprecipitation with an anti-YFP antibody and detected by immunoblot. The ratios of ubiquitinated over total YFP-tagged Amot130 pixel intensities are given. C, FLAG-tagged Amot130, AmotL1, or AmotL2 were co-expressed with HA-tagged ubiquitin and with Myc-tagged AIP4 or control vector. Immunoblot of AIP4 in lysates (anti-Myc) or immunoprecipitated FLAG-tagged total (anti-FLAG) or ubiquitinated (anti-HA) Amot family members were detected by immunoblot (IB). An alignment of the protein sequence encompassing Lys-481 in Amot with AmotL1 and AmotL2 is shown below this blot.