Characterization of 19S-associated deubiquitylating enzyme USP14 and UCHL5 in MM. (A) Purified CD138⁺ patient MM cells were lysed in protein lysis buffer and then subjected to immunoblotting with USP14 and UCHL5 antibodies. (B) MM cell lines and PBMCs from healthy donors were lysed in lysis buffer; lysates were then subjected to immunoblotting with USP14 and UCHL5 antibodies. Blots shown are representative of 3 independent experiments. (C) IHC analysis of bone marrow biopsies from normal donors and MM patients showing USP14 and UCHL5 expression. Scale bar = 60 µM. Red arrowheads indicate USP14 or UCHL5-positive cells (brown). (D) MM.1S cells were transfected with scramble siRNA, USP14 siRNA, UCHL5 siRNA, or USP14 plus UCHL5 siRNA by using the cell line Nucleofector Kit V. The cells were harvested 24 hours posttransfection and subjected to immunoblotting with USP14 and UCHL5 antibodies. (E) MM.1S cells were transfected with scramble siRNA, USP14 siRNA, UCHL5 siRNA, or both USP14 and UCHL5 siRNA, followed by viability analysis using a CellTiter-Glo assay. Percentage of cell viability was normalized to scramble siRNA control.