Angiopoietin system expression by HMCLs. Ang-1, Ang-2, Tie2, and β₂-microglobulin mRNA expression by RPMI 8226, OPM-2, U266, XG-1, XG-6, and the EBV⁺ cell line ARH-77 were evaluated by RT-PCR. (A) Leukemia cell line K562 was used as positive control (CON+), and CD19+ from healthy subjects was used as negative control for all factors. (B-C) Ang-1 protein expression and secretion by HMCLs were checked by Western blot analysis and immunoprecipitation, respectively. Conditioned medium of XG-6 was incubated with 0.5 μg anti–Ang-1 antibody for 1 hour at 4°C. Then 20 μL Protein G PLUS-Agarose was added and incubated, with mixing for 2 hours at 4°C. After washing, immunoprecipitated material was recovered by boiling in sample loading buffer and was separated by electrophoresis. Silver staining was performed to detect the presence of soluble Ang-1 (B). XG-6 and RPMI 8226 (5 × 10⁶) were incubated in the presence or absence of IL-6 (20 ng/mL) or VEGF (10 ng/mL) for 48 hours, and cell lysates were evaluated for Ang-1 expression by Western blot analysis, as described in “Patients, materials, and methods” (C). (D) Ang-1 and Ang-2 immunostaining in K562 (positive control) and in HMCLs. Ang-1 immunostaining was performed in U266 and K562 using the indirect immunoperoxidase method/DAB or in XG-6 and RPMI 8226 using the alkaline phosphatase method/new fuscin, as described in “Patients, materials, and methods.” Ang-2 immunostaining was performed in RPMI 8226 and K562 using the indirect immunoperoxidase method/DAB. Original magnification, × 100.