American Society of Hematology

Figure 1

$Figure 1. STAT3 is constitutively phosphorylated on serine 727 residues in CLL cells. (A) Cell lysates of patients with CLL (CLL 1-7) and of A549 cells (control) were analyzed by Western immunoblotting using antiserine pSTAT3, antityrosine pSTAT3, or anti-STAT3 antibodies. (B) Cell lysates of CLL patient 3 were incubated with rabbit anti–human STAT3 antibodies for immunoprecipitation (I.P.) with protein A-agarose beads. Incubation of cell lysates with beads only was used as a negative control (beads). The immunoprecipitate was analyzed by Western immunoblotting using antiserine pSTAT3, antityrosine pSTAT3, or anti-pSTAT3 antibodies. 3T3 cells were used as a positive control (Cont.). (C) PB cells of CLL patient 8 were fractioned using immunomagnetic beads. CD19+ and CD19− cells were collected. Normal PB CD19+/CD20+ B cells were purified by immunomagnetic beads. Expression of serine pSTAT3 in CLL low-density cells (LDC), CD19+, and CD19− cells and normal donor B cells (B1 and B2) was analyzed by Western immunoblotting using antiserine pSTAT3, anti-STAT3, or anti–β-actin (loading control) antibodies. (D) CLL cells (CLL 9) were stimulated with IL-6 (50 ng/mL, 30 minutes) or left untreated. The cells were harvested at different time points after IL-6 stimulation. Cell lysates were analyzed by Western immunoblotting using antityrosine pSTAT3, antiserine pSTAT3, or anti-STAT3 antibodies. (E) CLL cells (CLL 10) were cultured in medium for 12 to 72 hours, the cells were harvested, and cell lysates were analyzed by Western immunoblotting using antiserine pSTAT3 or anti-STAT3 antibodies. Vertical lines indicate realignment of the same gel's image.$

STAT3 is constitutively phosphorylated on serine 727 residues in CLL cells. (A) Cell lysates of patients with CLL (CLL 1-7) and of A549 cells (control) were analyzed by Western immunoblotting using antiserine pSTAT3, antityrosine pSTAT3, or anti-STAT3 antibodies. (B) Cell lysates of CLL patient 3 were incubated with rabbit anti–human STAT3 antibodies for immunoprecipitation (I.P.) with protein A-agarose beads. Incubation of cell lysates with beads only was used as a negative control (beads). The immunoprecipitate was analyzed by Western immunoblotting using antiserine pSTAT3, antityrosine pSTAT3, or anti-pSTAT3 antibodies. 3T3 cells were used as a positive control (Cont.). (C) PB cells of CLL patient 8 were fractioned using immunomagnetic beads. CD19⁺ and CD19⁻ cells were collected. Normal PB CD19⁺/CD20⁺ B cells were purified by immunomagnetic beads. Expression of serine pSTAT3 in CLL low-density cells (LDC), CD19⁺, and CD19⁻ cells and normal donor B cells (B1 and B2) was analyzed by Western immunoblotting using antiserine pSTAT3, anti-STAT3, or anti–β-actin (loading control) antibodies. (D) CLL cells (CLL 9) were stimulated with IL-6 (50 ng/mL, 30 minutes) or left untreated. The cells were harvested at different time points after IL-6 stimulation. Cell lysates were analyzed by Western immunoblotting using antityrosine pSTAT3, antiserine pSTAT3, or anti-STAT3 antibodies. (E) CLL cells (CLL 10) were cultured in medium for 12 to 72 hours, the cells were harvested, and cell lysates were analyzed by Western immunoblotting using antiserine pSTAT3 or anti-STAT3 antibodies. Vertical lines indicate realignment of the same gel's image.

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