Figure 7
Figure 7. Constitutive BLyS/BR3 signaling activates NIK-induced NF-κB pathway activation through induction of TRAF3 degradation in DLBCL. (A) Cytoplasmic cell lysates from MS-GCB or HB-ABC DLBCL cells were immunoprecipitated with BR3 or TRAF3 antibodies and probed with TRAF3 or BR3 antibodies. IgG was used as a negative control. (B) DLBCL-HB cells were co-stained with TRAF3 (FITC, green) or BR3 (Texas Red, red) antibody and analyzed by confocal microscopy. Yellow color indicates co-localization of BR3 and the other probed proteins. (C) Total cell lysates from DLBCL cells (MS) transfected with specific BR3 shRNA or control shRNA were probed with TRAF3 or BR3 antibodies by Western blot analysis. Actin was used as a loading control. Relative protein level of each target molecule was measured with ImageJ densitometer software and normalized to the actin level. (D) Total cell lysates from normal Go B cells stimulated with or without human recombinant BLyS ligand were probed with TRAF3 antibody by Western blot analysis. Actin was used as a loading control. Relative protein level of each target molecule was measured with ImageJ densitometer software and normalized to the actin level. (E) Cytoplasmic extract from DLBCL cells (MS) was subjected to immunoprecipitation analysis with BR3 antibody and then probed with TRAF2, TRAF3, c-IAP1, c-IAP2, or BR3 antibodies by Western blot analysis. (F) DLBCL cells (HB) were co-stained with BR3 (Texas Red, red), along with TRAF2 (FITC, green), c-IAP1 (FITC, green), or c-IAP2 (FITC, green) antibodies, and then analyzed by confocal microscopy. Yellow color indicates co-localization of BR3 and the other probed proteins. (G) Cytoplasmic extracts purified from DLBCL cells (MS) treated with BLyS antibody or control cells were probed with TRAF3 BR3 or NIK antibody in Western blot analysis (left). These samples were also subjected to immunoprecipitation analysis with BR3 antibodies and then probed with TRAF3 or BR3 antibodies for Western blot analysis (right). (H) Cell lysates purified from DLBCL-MS cells transfected with c-IAP1, c-IAP2 shRNA, or control shRNA were analyzed by Western blot analysis with c-IAP1, c-IAP2, or TRAF3 antibody. Actin was used as loading control. Relative protein level of each target molecule was measured with ImageJ densitometer software and normalized to the actin level.

Constitutive BLyS/BR3 signaling activates NIK-induced NF-κB pathway activation through induction of TRAF3 degradation in DLBCL. (A) Cytoplasmic cell lysates from MS-GCB or HB-ABC DLBCL cells were immunoprecipitated with BR3 or TRAF3 antibodies and probed with TRAF3 or BR3 antibodies. IgG was used as a negative control. (B) DLBCL-HB cells were co-stained with TRAF3 (FITC, green) or BR3 (Texas Red, red) antibody and analyzed by confocal microscopy. Yellow color indicates co-localization of BR3 and the other probed proteins. (C) Total cell lysates from DLBCL cells (MS) transfected with specific BR3 shRNA or control shRNA were probed with TRAF3 or BR3 antibodies by Western blot analysis. Actin was used as a loading control. Relative protein level of each target molecule was measured with ImageJ densitometer software and normalized to the actin level. (D) Total cell lysates from normal Go B cells stimulated with or without human recombinant BLyS ligand were probed with TRAF3 antibody by Western blot analysis. Actin was used as a loading control. Relative protein level of each target molecule was measured with ImageJ densitometer software and normalized to the actin level. (E) Cytoplasmic extract from DLBCL cells (MS) was subjected to immunoprecipitation analysis with BR3 antibody and then probed with TRAF2, TRAF3, c-IAP1, c-IAP2, or BR3 antibodies by Western blot analysis. (F) DLBCL cells (HB) were co-stained with BR3 (Texas Red, red), along with TRAF2 (FITC, green), c-IAP1 (FITC, green), or c-IAP2 (FITC, green) antibodies, and then analyzed by confocal microscopy. Yellow color indicates co-localization of BR3 and the other probed proteins. (G) Cytoplasmic extracts purified from DLBCL cells (MS) treated with BLyS antibody or control cells were probed with TRAF3 BR3 or NIK antibody in Western blot analysis (left). These samples were also subjected to immunoprecipitation analysis with BR3 antibodies and then probed with TRAF3 or BR3 antibodies for Western blot analysis (right). (H) Cell lysates purified from DLBCL-MS cells transfected with c-IAP1, c-IAP2 shRNA, or control shRNA were analyzed by Western blot analysis with c-IAP1, c-IAP2, or TRAF3 antibody. Actin was used as loading control. Relative protein level of each target molecule was measured with ImageJ densitometer software and normalized to the actin level.

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