ΔCD20 protein expression. (A) Confocal microscopy analysis of wt and ΔCD20 protein expression, on 293T cells transfected with different constructs carrying flCD20, ΔCD20, or mutCD20 cDNA fused with a GFP sequence leading to the expression of a CD20/GFP fusion protein. Cells were imaged using a Fluoview FV1000 (Olympus) and were stained with DAPI (blue) for nuclear staining and also with either monoclonal anti-CD20 antibody (recognizing wt and mutCD20 forms; red), C-term anti-CD20 antibody (recognizing all CD20 forms; orange), or by GFP 385-nm excitation (green). wtCD20 and mutCD20-transfected cells show membrane staining according to the presence of the 4 transmembrane domains allowing anchoring, whereas ΔCD20/GFP staining is localized mainly within the cytoplasm and absent within the membrane. Simultaneous staining (intracellular and membrane) was achieved with an anti–C-term CD20 antibody. DIC indicates differential interference contrast; and ORF, open reading frame. Untransduced cells were used as controls. (B) Western blot (WB) analysis, after denaturing acrylamide electrophoresis, with anti–C-term CD20 antibody of whole-cell lysates from B- and T-cell lines (left) and a retrovirally transduced Raji cell line with a vector carrying the ΔCD20 cDNA (right). As expected, we detected a signal at position 33 to 35 kDa, corresponding to the wtCD20 protein isoforms (differentially phosphorylated), but also 2 additional bands at 15 to 17 kDa, corresponding to the size of the translated spliced mRNA. The 2 bands at position 15 to 17 kDa could correspond to different phosphorylation states of the ΔCD20 protein. Moreover, detection of an increased signal at the same size length after CD20 transduction confirmed that the smaller band is the product of the ΔCD20 mRNA translation. Antiactin WB on the whole-cell lysates was performed as controls.