N-terminal cleaved pyrin activates NF-κB and interacts with p65. (A) NF-κB DNA-binding activity was measured by EMSA in nuclear lysates prepared from HeLa cells that had been transfected with myc-tagged empty vector, N330, or C330, and incubated with or without TNF-α (12.5 ng/mL) for 1 hour. As indicated, either unlabeled excess NF-κB wt (WT-cold) or mutant (MT-cold) oligonucleotides were added to the EMSA reactions as a competitor to assay binding specificity. (B) HeLa cells were cotransfected with the indicated pyrin constructs and either p65 or p50. From the nuclear lysates, NF-κB DNA-binding activity was measured by EMSA as in panel A. (C) Myc-tagged p50 (left panels) or p65 (right panels) was cotransfected into PT67 cells with GST vector, GST-fused pyrin, GST-N330, or GST-C330. Lysates were subjected to GST pull-down assay followed by Western blotting with antimyc Ab (top panels). Expression of transfected constructs is shown in the bottom 2 panels (cell lysates) probed with antimyc or anti-GST Abs. (D) Lysates from PBMCs of a healthy donor were immunoprecipitated with anti-p65 Ab or control IgG. Cell lysates and eluted proteins were analyzed by Western blotting with antipyrin (top) or anti-p65 Abs (bottom). Data are from a representative experiment from 3 separate subjects. (E) U937 cells were transduced with retroviral constructs expressing N330-myc or with empty vector. After G418 selection, cell lysates were immunoprecipitated with anti-p65 Ab or control IgG, and analyzed as in panel D. (F) HeLa cells were transfected with V5-tagged p65 (p65-V5) alone (i-ii), or cotransfected with p65-V5 and N330-myc (iii-vi), or with p65-V5 and myc-tagged full-length pyrin (vii-xi). After 24 hours, p65 and pyrin variants were stained with AlexaFluor 488–conjugated anti-V5 Ab (green) and AlexaFluor 568–conjugated antimyc Ab (red), respectively. Cells were counterstained with DAPI (blue) and visualized on a Leica DMR microscope. Expressed p65 is shown in the first column (i, iii, and vii); pyrin variants are shown in the second column (iv and viii); merged images for p65 and pyrin variants are in the third column (v and ix); merged images for p65, pyrin variants, and nuclei are in the fourth column (ii, vi, and x). (G) IκB-α–V5, p65-V5, and N330-myc were transfected individually, or cotransfected as indicated into HeLa cells. After 24 hours, total cell lysates (top 2 panels) and nuclear extracts (bottom 2 panels) were prepared and subjected to SDS-PAGE for Western blotting with anti-V5 Ab (top panel and third panel), anti–β-actin Ab (for loading control of whole-cell extracts), or anti-Ran Ab (for loading control of nuclear extracts). Asterisk denotes degraded 30-kDa IκB-α fragment that is explained in “N330 interacts with p65 and enhances the nuclear localization of p65.”