Stabilization of SCF^Skp2 substrates by CpdA in multiple myeloma cells. (A) Extracts from DMSO- or CpdA-treated MM.1S MM cells were used in the previously detailed p27 in vitro ubiquitination assay to detect inhibition of SCF^Skp2 E3 ligase function (left panel). Image J software measurement showed that CpdA was again able to inhibit p27 ubiquitination in a dose-dependent fashion in MM1.S cells, with 10-, 20-, and 25-μM concentrations resulting in a reduction of 8%, 44%, and 51% compared with controls. Addition of excess components of SCF^Skp2 restored ubiquitination (middle panel), but there was no effect on the inhibition of p27 ubiquitination by adding exogenous Cks1 (right panel). (B) RPMI 8226 cells treated with CpdA were then examined for the content of several SCF substrate proteins and cell cycle–related proteins by Western blotting. (C) RPMI 8226 cells were treated with DMSO, CpdA, or bortezomib for 24 hours. Whole-cell lysates were subjected to Western blotting using anti–HSP-27, phospho–HSP-27, HSP-70, and HSP-90 antibodies.