Figure 2
Figure 2. Activation of NF-κB in CLL B cells by rhBAFF or rhAPRIL. (A) Immunoblot analyses with anti-p100 or anti-p65 antibodies. CLL B cells were cultured with or without rhBAFF (50 ng/mL) or rhAPRIL (500 ng/mL) for 24 hours. Cytoplasmic and nuclear extracts were prepared as described in “Materials and methods.” We evaluated for equal loading in each lane by stripping the blot and probing it with antibodies specific for β-actin (for cytoplasmic extracts) or SP-1 (for nuclear extracts). The MFIR for each receptor on this CLL sample was 2.8, 3.6, or 31.3 for BCMA, TACI, or BR3, respectively. Translocation of p65 to the nucleus was seen in CLL cells treated with rhBAFF or rhAPRIL. In contrast, translocation of p52 was observed only in CLL cells treated with rhBAFF. (B) Immunoblot analysis with anti-IκBα antibodies. CLL B cells were cultured with or without rhBAFF (50 ng/mL) or rhAPRIL (500 ng/mL) for 30 minutes. Total cell lysates were prepared as described in “Materials and methods.” Degradation of IκBα was observed in CLL cells treated with rhBAFF or rhAPRIL. (C) EMSAs of nuclear extracts from CLL cells. CLL B cells were cultured with or without rhBAFF (50 ng/mL), rhAPRIL (500 ng/mL), BCMA-Fc (10 μg/mL), BR3-Fc (10 μg/mL), anti-BR3 (10 μg/mL), or control immunoglobulin (10 μg/mL) for 24 hours. We monitored for equal loading of protein in each lane by examining NF-Y binding to DNA. The MFIR of CLL cells stained for BCMA, TACI, or BR3 was 3.7, 8.8, or 24.3, respectively. Nuclear extracts prepared from CLL cells cultured with rhBAFF or rhAPRIL contained increased amounts of proteins capable of binding the NF-κB consensus motifs, which experienced a supershift when preincubated with anti-p50 or anti-p65 antibodies. Nuclear extracts of CLL cells treated with rhBAFF or rhAPRIL in the presence of BCMA-Fc or BR3-Fc had less NF-κB binding activity. However, nuclear extracts of CLL cells treated with rhBAFF and anti-BR3 antibody contained amounts of NF-κB binding factors similar to those of extracts prepared from CLL cells treated with rhBAFF alone.

Activation of NF-κB in CLL B cells by rhBAFF or rhAPRIL. (A) Immunoblot analyses with anti-p100 or anti-p65 antibodies. CLL B cells were cultured with or without rhBAFF (50 ng/mL) or rhAPRIL (500 ng/mL) for 24 hours. Cytoplasmic and nuclear extracts were prepared as described in “Materials and methods.” We evaluated for equal loading in each lane by stripping the blot and probing it with antibodies specific for β-actin (for cytoplasmic extracts) or SP-1 (for nuclear extracts). The MFIR for each receptor on this CLL sample was 2.8, 3.6, or 31.3 for BCMA, TACI, or BR3, respectively. Translocation of p65 to the nucleus was seen in CLL cells treated with rhBAFF or rhAPRIL. In contrast, translocation of p52 was observed only in CLL cells treated with rhBAFF. (B) Immunoblot analysis with anti-IκBα antibodies. CLL B cells were cultured with or without rhBAFF (50 ng/mL) or rhAPRIL (500 ng/mL) for 30 minutes. Total cell lysates were prepared as described in “Materials and methods.” Degradation of IκBα was observed in CLL cells treated with rhBAFF or rhAPRIL. (C) EMSAs of nuclear extracts from CLL cells. CLL B cells were cultured with or without rhBAFF (50 ng/mL), rhAPRIL (500 ng/mL), BCMA-Fc (10 μg/mL), BR3-Fc (10 μg/mL), anti-BR3 (10 μg/mL), or control immunoglobulin (10 μg/mL) for 24 hours. We monitored for equal loading of protein in each lane by examining NF-Y binding to DNA. The MFIR of CLL cells stained for BCMA, TACI, or BR3 was 3.7, 8.8, or 24.3, respectively. Nuclear extracts prepared from CLL cells cultured with rhBAFF or rhAPRIL contained increased amounts of proteins capable of binding the NF-κB consensus motifs, which experienced a supershift when preincubated with anti-p50 or anti-p65 antibodies. Nuclear extracts of CLL cells treated with rhBAFF or rhAPRIL in the presence of BCMA-Fc or BR3-Fc had less NF-κB binding activity. However, nuclear extracts of CLL cells treated with rhBAFF and anti-BR3 antibody contained amounts of NF-κB binding factors similar to those of extracts prepared from CLL cells treated with rhBAFF alone.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal