The influence of bortezomib on the expression of CD20 in Raji cells. (A) Raji cells were incubated with either diluent or bortezomib (10-50nM) for 12, 24, or 48 hours. Then, cells (10⁵/100 μL) were incubated with saturating amounts of FITC-conjugated mAb against CD20 for 30 minutes on ice in the dark and analyzed in a FACSCalibur using CellQuest Pro Software Version 5.2. (B) CD20 protein levels in Raji cells incubated with either diluent or 20 and 50nM bortezomib for 12, 24, or 48 hours were assayed with Western blotting (NCL-CD20-L26 mAb). (C) CD20 mRNA levels in Raji cells incubated with either diluent or 20 and 50nM bortezomib for 12, 24, or 48 hours were assayed with RT-PCR. (D) Raji cells (10⁵/100 μL) were incubated with either diluent or bortezomib (10-50nM) for 12, 24, or 48 hours and stained for CD20, CD21, CD45RA, CD54, or HLA-DR. The mean fluorescence intensity (MFI) serves as a measure for mAb binding on a per-cell basis. *P < .05 (Student t test). (E) Primary CD20⁺ tumor cells (chronic lymphocytic leukemia: patient 1 [Pt1], diffuse large B-cell lymphoma: Pt2, and mantle cell lymphoma: Pt3) were incubated with either 1 or 10 nM bortezomib for 24 or 48 hours. Then, cells (10⁵/100 μL) were incubated with saturating amounts of FITC-conjugated mAb against CD20 for 30 minutes on ice in the dark and analyzed in a FACSCalibur using CellQuest Pro Software Version 5.2. *P < .05 (Student t test). (F) Immunoprecipitation of Raji cell culture supernatants with anti-CD20 mAb (NCL-CD20-L26). Positive controls included a lane with CD20 immunoprecipitated from Raji lysates (L) and soluble recombinant HSP70 added at 20-ng/mL concentration to the cultured cells and immunoprecipitated with anti-HSP70 mAb (Sup). Lane designated rHSP70 refers to 20 ng of electrophoresed recombinant HSP70.