Figure 6
Figure 6. Nuclear BAFF-R interacts with NF-κB/c-Rel and functions as a transcription factor. (A) Comparison of luciferase activity in NHL-B (LBCL-MS) cells transfected with a pBind control vector, a pBindBAFF-R vector, both pBindBAFF-R and c-Rel expression vectors, or both pBindBAFF-R and p65 expression vectors. The error bars indicate SD of triplicate samples. (B) c-Rel protein was stained with Cy2 fluorescence; BAFF-R protein was stained with Cy3 fluorescence in the NHL-B-cell line (MS). Samples were analyzed by confocal microscope. TOPRO-3 was used as a nuclear marker. (C) The above experiment was repeated in representative biopsy-derived LBCL and MCL patient samples, as shown. (D) his-BAFF-R fusion protein was purified by MagneHis purification system from whole-cell lysates of NHL-B cells (MS) transfected with a pcDNA3.1-his-BAFF-R expression plasmid. Samples were probed with BAFF-R antibody, c-Rel antibody, HMG1 antibody, and his antibody (top panel) in WB. Wild-type BAFF-R protein complex was precipitated by BAFF-R antibody from the nuclear extract of NHL-B cells (MS) and probed with BAFF-R or c-Rel antibody in WB (bottom panel). Lamin B (a nuclear marker), syndecan 4 (a plasma membrane marker), and calreticulin (an endoplasmic reticulum marker) were used as loading control. (E) FRET analysis was performed in NHL-B cells (MS) cotransfected with GFP-BAFF-R fusion protein expression plasmid and dsRed–c-Rel fusion protein expression plasmid. Images of a representative transfected cell were taken with a confocal microscope before photobleaching (labeled “pre”) and after photobleaching (labeled “post”). (F) The pACT-c-Rel and pBIND-BAFF-R fusion vectors were cotransfected with pG5luc into NHL-B cells. Firefly and luciferase activity was measured and normalized by β-gal luciferase activity. The error bars indicate SD of triplicate samples. (G) ChIP analysis was performed in NHL-B (LBCL-MS) cells after precipitation of the protein-DNA complex with BAFF-R antibody. PCR analysis to detect the BLyS, CD154, Bfl-1/A1, Bcl-xL, and IL-8 promoters was performed using immunoprecipitated DNA, using proximal primers near the c-Rel binding region in these gene promoters. Actin was used as a control. (H) Whole-cell lysates from NHL-B cells transfected with control (empty) plasmid, a BAFF-R expression plasmid, or a BAFF-R NLS-mutant expression plasmid were blotted with BLyS, CD154, BCL-xL, Bfl-1/A1, or actin (loading control) antibody in WB.

Nuclear BAFF-R interacts with NF-κB/c-Rel and functions as a transcription factor. (A) Comparison of luciferase activity in NHL-B (LBCL-MS) cells transfected with a pBind control vector, a pBindBAFF-R vector, both pBindBAFF-R and c-Rel expression vectors, or both pBindBAFF-R and p65 expression vectors. The error bars indicate SD of triplicate samples. (B) c-Rel protein was stained with Cy2 fluorescence; BAFF-R protein was stained with Cy3 fluorescence in the NHL-B-cell line (MS). Samples were analyzed by confocal microscope. TOPRO-3 was used as a nuclear marker. (C) The above experiment was repeated in representative biopsy-derived LBCL and MCL patient samples, as shown. (D) his-BAFF-R fusion protein was purified by MagneHis purification system from whole-cell lysates of NHL-B cells (MS) transfected with a pcDNA3.1-his-BAFF-R expression plasmid. Samples were probed with BAFF-R antibody, c-Rel antibody, HMG1 antibody, and his antibody (top panel) in WB. Wild-type BAFF-R protein complex was precipitated by BAFF-R antibody from the nuclear extract of NHL-B cells (MS) and probed with BAFF-R or c-Rel antibody in WB (bottom panel). Lamin B (a nuclear marker), syndecan 4 (a plasma membrane marker), and calreticulin (an endoplasmic reticulum marker) were used as loading control. (E) FRET analysis was performed in NHL-B cells (MS) cotransfected with GFP-BAFF-R fusion protein expression plasmid and dsRed–c-Rel fusion protein expression plasmid. Images of a representative transfected cell were taken with a confocal microscope before photobleaching (labeled “pre”) and after photobleaching (labeled “post”). (F) The pACT-c-Rel and pBIND-BAFF-R fusion vectors were cotransfected with pG5luc into NHL-B cells. Firefly and luciferase activity was measured and normalized by β-gal luciferase activity. The error bars indicate SD of triplicate samples. (G) ChIP analysis was performed in NHL-B (LBCL-MS) cells after precipitation of the protein-DNA complex with BAFF-R antibody. PCR analysis to detect the BLyS, CD154, Bfl-1/A1, Bcl-xL, and IL-8 promoters was performed using immunoprecipitated DNA, using proximal primers near the c-Rel binding region in these gene promoters. Actin was used as a control. (H) Whole-cell lysates from NHL-B cells transfected with control (empty) plasmid, a BAFF-R expression plasmid, or a BAFF-R NLS-mutant expression plasmid were blotted with BLyS, CD154, BCL-xL, Bfl-1/A1, or actin (loading control) antibody in WB.

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