American Society of Hematology

Figure 5.

$Figure 5. Activation of NF-κB in CLL B cells by rhBAFF or rhAPRIL. (A) Processing of p100 and nuclear translocation of p52 or p65. CLL B cells were cultured with or without SDF-1α (500 ng/mL), rhBAFF (50 ng/mL), and rhAPRIL (500 ng/mL) for 24 hours. Cytoplasmic and nuclear extracts were prepared as described in “Materials and methods” for immunoblot analysis with anti-p100 or anti-p65 antibodies as indicated on the left side of each panel. The agent used to treat the CLL cells is indicated at the top of each panel under the label indicating whether the extract was derived from cytoplasmic (left panel) or nuclear (right panel) cell fractions. We evaluated for equal loading in each lane by stripping the blot and probing it again with antibodies specific for β-actin (for cytoplasmic extracts) or SP-1 (for nuclear extracts), as indicated on the left side of each panel. (B) Degradation of IκBα. Extracts of CLL cells were prepared for immunoblot analysis prior to treatment (Pre-Tx) or after a 30-minute incubation with culture medium alone (Medium) or medium supplemented with SDF-1α (500 ng/mL), rhBAFF (50 ng/mL), rhAPRIL (500 ng/mL), or TNFα (50 ng/mL), as indicated at the top of each lane. The immunoblot was probed with antibodies specific for IκBα (top blot). We evaluated for equal loading in each lane by stripping the blot and probing it again with antibodies specific for β-actin (bottom blot).$

Activation of NF-κB in CLL B cells by rhBAFF or rhAPRIL. (A) Processing of p100 and nuclear translocation of p52 or p65. CLL B cells were cultured with or without SDF-1α (500 ng/mL), rhBAFF (50 ng/mL), and rhAPRIL (500 ng/mL) for 24 hours. Cytoplasmic and nuclear extracts were prepared as described in “Materials and methods” for immunoblot analysis with anti-p100 or anti-p65 antibodies as indicated on the left side of each panel. The agent used to treat the CLL cells is indicated at the top of each panel under the label indicating whether the extract was derived from cytoplasmic (left panel) or nuclear (right panel) cell fractions. We evaluated for equal loading in each lane by stripping the blot and probing it again with antibodies specific for β-actin (for cytoplasmic extracts) or SP-1 (for nuclear extracts), as indicated on the left side of each panel. (B) Degradation of IκBα. Extracts of CLL cells were prepared for immunoblot analysis prior to treatment (Pre-Tx) or after a 30-minute incubation with culture medium alone (Medium) or medium supplemented with SDF-1α (500 ng/mL), rhBAFF (50 ng/mL), rhAPRIL (500 ng/mL), or TNFα (50 ng/mL), as indicated at the top of each lane. The immunoblot was probed with antibodies specific for IκBα (top blot). We evaluated for equal loading in each lane by stripping the blot and probing it again with antibodies specific for β-actin (bottom blot).

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