Proteasome inhibition increases ubiquitination of CD20. (A) Raji cells were seeded into the flask bottles at a concentration of 2 × 106 cells/10 mL. The cells were exposed to bortezomib at indicated concentrations for 24 or 48 hours, after which protein lysates were prepared. CD20 antigen was immunoprecipitated from samples with protein G bead slurry and subjected to Western blot analysis. Blots were sequentially probed (after stripping) with antiubiquitin and anti-CD20 antibodies. IP indicates immunoprecipitation; WB, Western blotting. (B) To assess the ubiquitination of membrane CD20, Raji cells were seeded into the flask bottles at a concentration of 2 × 106 cells/10 mL. The cells were exposed to bortezomib at indicated concentrations for 24 or 48 hours. Then, extracellular portions of membrane proteins were bound with EZ-link sulfo-NHS-biotin. Labeled cells were lysed and biotinylated proteins were precipitated with immobilized NeutrAvidin protein. Subsequently, from the mixture of boiled precipitated surface proteins, an additional immunoprecipitation step with anti-CD20 mAb (NCL-CD20-L26) was performed. Immunoprecipitates underwent electrophoresis followed by blotting with antiubiquitin and anti-CD20 antibodies. ICAM-1 is a marker for membrane fraction; and tubulin, for a cytosolic fraction. L indicates the total cell lysate that served as a positive control; WB, Western blotting.
