Figure 7.
Figure 7. Role of TRAF6 and NIK in RANKL signaling. (A) Dominant-negative TRAF6 and TRAF2, but not TRAF5, suppress RANKL-induced NF-κB reporter gene expression in p60-/-p80-/- macrophages. 0.5 × 106 cells were transiently transfected with a NF-κB-containing plasmid alone or with indicated plasmids for 24 hours. After transfection, cells were washed and treated with 10 nM RANKL for an additional 24 hours. The supernatants of the culture medium were assayed for SEAP activity as described in “Materials and methods.” (B) TRAF6-binding protein suppresses RANKL-induced NF-κB activation in p60-/-p80-/- macrophages. 1.0 × 106 cells were pretreated with the indicated concentrations of TRAF6-binding protein (TRAF6-BP), 300 μM TRAF6-free, or 300 μM TRAF6-BP mutant peptide and then treated with 5 nM RANKL for 30 minutes. Nuclear extracts were prepared and analyzed for NF-κB activation by EMSA as described in “Materials and methods.” (C) Deletion of TNF receptors potentiates recruitment of TRAF6 into RANK complex. 1.0 × 107 cells were treated with 10 nM RANKL for 15 minutes; whole-cell extracts were prepared, incubated with anti-RANK antibody for 2 hours, and then immunoprecipitated with protein A/G-Sepharose beads. The beads were washed and resolved by 10% SDS-PAGE and electrotransferred to a nitrocellulose membrane. Western blot analysis was performed using anti-TRAF6 antibody. Fifty micrograms of the same protein extract was resolved by 10% SDS-PAGE and electrotransferred to a nitrocellulose membrane. Western blot analysis was performed using anti-TRAF6 antibody. (D) Deletion of TNF receptors potentiates association of NIK to TRAF6. 1.0 × 107 cells were either untreated or treated with 10 nM RANKL for 15 minutes; whole-cell extracts were prepared, incubated with anti-TRAF6 antibody for 2 hours, and then immunoprecipitated with protein A/G-Sepharose beads. The beads were washed and resolved by 7.5% SDS-PAGE and electrotransferred to a nitrocellulose membrane. Western blot analysis was performed using anti-NIK antibody. The same membrane was also blotted with anti-TRAF6 antibody.

Role of TRAF6 and NIK in RANKL signaling. (A) Dominant-negative TRAF6 and TRAF2, but not TRAF5, suppress RANKL-induced NF-κB reporter gene expression in p60-/-p80-/- macrophages. 0.5 × 106 cells were transiently transfected with a NF-κB-containing plasmid alone or with indicated plasmids for 24 hours. After transfection, cells were washed and treated with 10 nM RANKL for an additional 24 hours. The supernatants of the culture medium were assayed for SEAP activity as described in “Materials and methods.” (B) TRAF6-binding protein suppresses RANKL-induced NF-κB activation in p60-/-p80-/- macrophages. 1.0 × 106 cells were pretreated with the indicated concentrations of TRAF6-binding protein (TRAF6-BP), 300 μM TRAF6-free, or 300 μM TRAF6-BP mutant peptide and then treated with 5 nM RANKL for 30 minutes. Nuclear extracts were prepared and analyzed for NF-κB activation by EMSA as described in “Materials and methods.” (C) Deletion of TNF receptors potentiates recruitment of TRAF6 into RANK complex. 1.0 × 107 cells were treated with 10 nM RANKL for 15 minutes; whole-cell extracts were prepared, incubated with anti-RANK antibody for 2 hours, and then immunoprecipitated with protein A/G-Sepharose beads. The beads were washed and resolved by 10% SDS-PAGE and electrotransferred to a nitrocellulose membrane. Western blot analysis was performed using anti-TRAF6 antibody. Fifty micrograms of the same protein extract was resolved by 10% SDS-PAGE and electrotransferred to a nitrocellulose membrane. Western blot analysis was performed using anti-TRAF6 antibody. (D) Deletion of TNF receptors potentiates association of NIK to TRAF6. 1.0 × 107 cells were either untreated or treated with 10 nM RANKL for 15 minutes; whole-cell extracts were prepared, incubated with anti-TRAF6 antibody for 2 hours, and then immunoprecipitated with protein A/G-Sepharose beads. The beads were washed and resolved by 7.5% SDS-PAGE and electrotransferred to a nitrocellulose membrane. Western blot analysis was performed using anti-NIK antibody. The same membrane was also blotted with anti-TRAF6 antibody.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal