Truncated protein isoforms of CD19 provide proliferative advantage.

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Truncated protein isoforms of CD19 provide proliferative advantage. Truncated protein isoforms of CD19 provide proliferative advantage. A, CD19 proteins encoded by the full-length (FL) and the Δex2 and Δex5–6 isoforms of CD19 mRNA. The epitope recognized by CART-19 is encoded by a sequence contained within exons 1–4. The transmembrane domain is encoded by exons 5 and 6. B, immunoblotting for CD19 in protein lysates from xenografted tumor samples using antibodies recognizing the extracellular domain (clone 3F5 from Origene; top) or the cytosolic domain (Santa Cruz Biotechnologies; sc-69735; bottom) C, immunoblotting for CD19 in protein lysates from a panel of cell lines representing human B-cell malignancies. Arrows indicate full-length and the Δex2 isoforms. The antibody used (Santa Cruz Biotechnologies; sc-69735) recognizes the cytosolic domains. D, qRT-PCR analysis of CD19 mRNA splicing variants in NALM-6 that were treated with actinomycin D for indicated periods of time. MYC mRNA was used as internal control for effective inhibition of transcription. E, immunoblotting for CD19 in lysates from CD19-negative Myc5 murine B-lymphoid cells transduced with CD19 retroviral constructs. Arrows indicate full-length, Δex2, and Δex5–6 isoforms. F, immunoblotting analysis of CD19 protein stability in cells from E. Cultures were treated with cycloheximide for indicated periods of time. Labile MYC protein was used as control for effective inhibition of protein synthesis. G, flow cytometry performed on CD19-negative murine Myc5 cells infected with empty (black), full-length CD19 (red), or CD19 Δex2 (green) expressing retrovirus. H, growth rates of first three cultures from D. Average fold increase in cell numbers from triplicate plates is shown. Statistical significance per Student t test, with *, P ≤ 0.05 and **, P < 0.01. Elena Sotillo et al. Cancer Discov 2015;5:1282-1295 ©2015 by American Association for Cancer Research