Figure 1.
Schematic diagram of the experimental procedures.
The application of SILAC in differential phosphoproteomic profiling of myeloma cells U266 treated by bortezomib.
Figure 2.
Identification and quantization of Ser38 phosphorylation of stathmin.
(A) Representative MS/MS spectrum of phosphopeptide ESVPEFPLpSPPK. (B) Representative chromatogram of ESVPEFPLpSPPK. Ion chromatograms of [12C6] lysine- and [13C6] lysine-containing peptides identified by MS/MS were extracted from the series of MS scans by using MSQuant. (C) Western blot analyses using specific antibodies showed that phosphorylation of stathmin at Ser38 was increased, whereas steady-state stathmin remained almost unchanged upon bortezomib treatment.
Figure 3.
Bioinformatic analysis of identified DEPPs.
(A) Pie chart representations of the distribution of identified DEPPs according to their molecular functions. Categorizations were based on information provided by the online resource PANTHER classification system. (B) The protein interaction network of the identified DEPPs. The network was mapped using the STRING system (http://string.embl.de/) based on evidence with different types. Different line colors represent the types of evidence for the associations, which are shown in the legend.
Figure 4.
Phosphorylation pattern of stathmin under bortezomib treatment.
(A) Upregulation of the phosphorylation at Ser16 and Ser38 and down-regulation of the phosphorylation at Ser25 were observed in bortezomib-treated U266 cells. (B) Activation of kinases targeting these residues (CaMKII, CDK2, and MAPK) was studied by Western blot using specific antibodies. Equal protein loading was assessed with actin.
Figure 5.
Mutation of stathmin phosphorylation sites decreases sensitivity to bortezomib and influences tubulin polymerization.
(A) U266 cells were stably transfected with HA-tagged wild type (WT) and mutant (S16A, S25A, S38A) stathmin constructs or its empty vector (NC). (B) Cells were exposed to 3 nM bortezomib for 24 hr, after which the percentage of apoptotic cells was determined by Annexin V/PI staining and flow cytometry. Results represent the means (± SD) for 3 separate experiments performed in triplicate. (*P<0.05 and **P<0.01). (C) Analysis of tubulin polymerization in U266 or derived cells with or without bortezomib treatment. Lysates from these cells were obtained from cells either treated or not with bortezomib at the indicated concentration. Lysates were separated into polymerized (P) or soluble (S) fractions and aliquots of equal volume were separated by SDS-PAGE, the blots probed with anti α-tubulin and the percent of polymerized tubulin calculated for each ‘P’ and ‘S’ pair.
Table 1.
Percent of polymerized tubulin in U266 and derived cells treated with bortezomib.