Quantitative Phosphoproteomics of Proteasome Inhibition in Multiple Myeloma Cells
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.