American Society of Hematology

Figure 1

$Figure 1. CD40 interacts with c-Rel in the nucleus of LBCL cells. (A) Coimmunoprecipitation assays were performed with the nuclear extracts of LBCL-MS and LBCL-McA cells. Nuclear extracts (1 mg) from LBCL cells (MS and McA) were immunoprecipitated with a polyclonal CD40 antibody or IgG antiserum (negative control) respectively. Immunoprecipitated complexes were resolved on SDS–polyacrylamide gel electrophoresis (PAGE), and subjected to Western blotting with anti–c-Rel, anti-p65, anti–Oct-1, and anti–β-actin antibodies. Input indicates 25 μg nuclear extract; NE, nuclear extract. (B) MS cells were cotransfected with expression vectors for c-Rel (pCMV-c-Rel) and CD40wt (pcDNA3.1/myc/his-CD40). Forty-eight hours after transfection, the nuclear fraction was extracted and immunoprecipitated with monoclonal anti-Myc tag antibody. Immunoprecipitates were then Western blotted with anti-Myc (detecting recombinant CD40), anti–c-Rel, anti–Oct-1, and anti–β-actin antibodies. Input indicates 25 μg nuclear extract. (C) LBCL cells (MS) were fixed with methanol and stained for CD40 (green), c-rel (red), and nuclear marker Topro-3 (blue) and analyzed by confocal immunofluorescence analysis. Colocalization of CD40 and c-Rel appears yellow. See “Materials and methods, Confocal microscopic analysis” for image acquisition information.$

CD40 interacts with c-Rel in the nucleus of LBCL cells. (A) Coimmunoprecipitation assays were performed with the nuclear extracts of LBCL-MS and LBCL-McA cells. Nuclear extracts (1 mg) from LBCL cells (MS and McA) were immunoprecipitated with a polyclonal CD40 antibody or IgG antiserum (negative control) respectively. Immunoprecipitated complexes were resolved on SDS–polyacrylamide gel electrophoresis (PAGE), and subjected to Western blotting with anti–c-Rel, anti-p65, anti–Oct-1, and anti–β-actin antibodies. Input indicates 25 μg nuclear extract; NE, nuclear extract. (B) MS cells were cotransfected with expression vectors for c-Rel (pCMV-c-Rel) and CD40wt (pcDNA3.1/myc/his-CD40). Forty-eight hours after transfection, the nuclear fraction was extracted and immunoprecipitated with monoclonal anti-Myc tag antibody. Immunoprecipitates were then Western blotted with anti-Myc (detecting recombinant CD40), anti–c-Rel, anti–Oct-1, and anti–β-actin antibodies. Input indicates 25 μg nuclear extract. (C) LBCL cells (MS) were fixed with methanol and stained for CD40 (green), c-rel (red), and nuclear marker Topro-3 (blue) and analyzed by confocal immunofluorescence analysis. Colocalization of CD40 and c-Rel appears yellow. See “Materials and methods, Confocal microscopic analysis” for image acquisition information.

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