FIGURE 5.

Identification of ubiquitylated lysine residues in LigI, and the effect of replacing ubiquitylated lysine residues with arginine in LigI steady state levels in serum-starved cells. Anti-FLAG immunoprecipitates from nuclear extracts of labeled 293T cells expressing FLAG-tagged LigI mixed with nuclear extracts of unlabeled parental 293T cells were digested with trypsin and tryptic peptides identified by mass spectrometry as described under “Experimental Procedures.” a–c, MS/MS spectra of peptides bearing ubiquitylation of Lys-376 (a), Lys-79 (b), and Lys-192 (c) in LigI. K^#, ubiquitylation site. R′, [¹³C₆¹⁵N₄]arginine. Red peaks correspond to b ions (masses of fragments generated from the peptide N terminus), and blue peaks correspond to y ions (masses of fragments generated from the peptide C terminus). d, asynchronously proliferating GM00087 cells expressing the FLAG-tagged K79R,K192R,K226R,K376R version of LigI were serum-starved for the indicated times. FLAG-tagged and endogenous versions of LigI were detected by immunoblotting with the LigI antibody.