The activity of the ubiquitination pathway is increased in malignant cell lines and primary patient samples. (A) Total cellular proteins were isolated from leukemia cell lines, primary AML patient samples, and normal hematopoietic cells (PBSCs). Equal amounts of protein were analyzed by SDS-PAGE followed by immunoblotting with anti-ubiquitin, anti–α-tubulin, or anti-GAPDH antibodies (the latter as protein loading control). (B) Proteasome chymotrypsin-like (CT-L), caspase-like (C-L), and trypsin-like (T-L) activities were measured in primary AML (n = 12), primary normal hematopoietic cells (n = 6), and leukemia cell lines as described in “20S proteasome assays” with the use of cell lysates that were prepared from the samples that included those used for immunoblotting assays in panel A. Data represent the mean fold increase ± SD activity compared with the PBSC normal controls.