Rituximab binding increases CD20 ubiquitination. (A) Raji cells were seeded into the 25-cm2 bottles at a concentration of 5 × 105 cells/10 mL and exposed to 10 μg/mL rituximab for 24, 48, or 72 hours, after which protein lysates were prepared and analyzed in Western blotting. Each lane was loaded with 20 μg of total protein. Blots were sequentially probed (after stripping) with antitubulin and anti-CD20 antibodies. (B) Raji cells were seeded into the 25-cm2 bottles at a concentration of 5 × 105 cells/10 mL. The cells were exposed to 10 μg/mL rituximab for 24, 48, or 72 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.
