ΔCD20 alternative transcripts code for an intracellular ΔCD20 protein. (A) Site-directed mutagenesis was performed on the wtCD20 sequence, using the QuikChange II XL Site-Directed Mutagenesis Kit (Stratagene), according to manufacturer recommendations to kill the AS, with respect to the amino acid sequence. Left panel shows electropherograms after site-directed mutagenesis, confirming that the third nucleotide of the CAG codon (Gln) is replaced by an A nucleotide. Right panel shows cytometry detection of the mutCD20 protein with a monoclonal anti-CD20 antibody at the cell surface of transfected PG13 cells. (B) flCD20- or ΔCD20-PCR on DNA or cDNA of transfected PG13 packaging cell line with wtCD20, ΔCD20, or mutCD20 retroviral plasmids. Neo-PCR was performed to control cell transfection and hypoxanthine-guanine-phosphoribosyl transferase (HPRT)–PCR to confirm the absence of inhibitors of the PCR reactions. flCD20 and ΔCD20 plasmids were used as positive controls. The dashed box highlights the absence of ΔCD20 PCR products, even with the ΔCD20-specific PCR, after transfection of PG13 with the mut-CD20 construct. (C) Western blot analysis with the C-term anti-CD20 on cell lysates from the bulk cell population or isolated cloned PG13 cells transfected with constructs carrying wtCD20 or mutCD20 cDNA sequences. Absence of detection at 15 to 17 kDa confirms that the splice sequence is the source of the ΔCD20 protein expression. (D) Western blot analysis on whole protein lysate (W) or subcellular fractions as cytoplasm (C) and membrane (M) for 4 different B-cell lines using the C-term CD20 antibody.