Journal: The Journal of Experimental Medicine
Article Title: NFATC3 promotes IRF7 transcriptional activity in plasmacytoid dendritic cells
Figure Lengend Snippet: NFATC3 selectively binds to IRF7 and enhances its transcriptional activity. (A) Fresh isolated human primary pDCs or pDCs stimulated with CpG A for 4 h. Cells were fixed with paraformaldehyde, permeablized, stained with anti-IRF7 (green) and anti-NFATC3 (red), and mounted with Prolong gold antifade reagent with DAPI (blue). Cells were analyzed using confocal microscopy. (B) Untouched human pDCs were isolated as described in the Confocal microscopy section of Materials and methods. pDCs were left untreated or treated with 1 µM CpG A for 3 h, and pDCs were lysed and immunoprecipitated (IP) with NFATC3 antibody. IRF7 was detected by immunoblotting (IB). (C) Purified GST or GST-tagged NFATC3 were incubated with Myc-tagged IRF7. Myc-tagged IRF7 was immunoprecipitated with Myc–agarose beads. GST was detected by anti-GST HRP conjugate antibody. (D) Purified Myc-tagged IRF3 or IRF7 was incubated with GST-tagged NFATC3, and GST-tagged NFATC3 was immunoprecipitated with glutathione Sepharose beads. Then, Myc-tagged proteins were detected by anti-Myc antibody. (E) HEK-293T cells were transfected with HA-tagged NFAT plasmids and Myc-tagged IRF7; 36 h after transfection, cell lysates were immunoprecipitated with anti-HA beads, and then the immunoprecipitates and lysates were analyzed with anti-HA or anti-Myc antibodies. (F) HEK-293T cells were transfected with HA-tagged NFATC3 plasmids and Myc-tagged MyD88, IRF3, and IRF7 plasmids; 36 h after transfection, cell lysates were immunoprecipitated with anti-HA beads, and then the immunoprecipitates and lysates were analyzed with anti-HA or anti-Myc antibodies. (G and H) HEK-293T cells were transfected with 100 ng IFN-α4 luciferase reporter plasmid (IFNα4-Luc) or IFN-β luciferase reporter plasmid (IFNβ-Luc) and 0.5 ng IRF7 expression vector together with an increasing amount of NFATC3 expression vector (25, 50, and 100 ng). 0.1 ng renilla luciferase reporter plasmid was transfected simultaneously as an internal control. Results are presented as fold induction relative to the activity of renilla luciferase. (I) HEK-293T cells were transfected with 100 ng NF-κB luciferase reporter plasmid (NFκB-Luc) together with 5 ng MyD88 expression vector or an increasing amount of NFATC3 expression vector. (J) HEK-293T cells were transfected with 100 ng IFN-β luciferase reporter plasmid (IFNβ-Luc) together with 5 ng IKKi expression vector or an increasing amount of NFATC3 expression vector. (G–J) Data are representative of four independent experiments.
Article Snippet: The following antibodies were used for immunoblot analysis and/or immunoprecipitation: anti-IRF7 (sc-74472), anti-IRF7 (sc-9083), anti-NFATC1 (sc-13033), anti-NFATC2 (sc-7296), anti-NFATC3 (sc-8321), anti-NFATC4 (sc-13036), and anti-NFAT5 (sc-13035) were from Santa Cruz Biotechnology, Inc.; anti-IRF7 (51–3300) was from Thermo Fisher Scientific; anti-MyD88 (4283), anti–NF-κB1 p105/p50, and anti-HDAC1 (5356) were from Cell Signaling Technology; and anti-NFATC3 (A303-572A) was from Bethyl Laboratories, Inc. HRP-conjugated anti-HA (ab1265), anti-Myc (ab62928), and anti-transferrin receptor antibody (ab84036) were from Abcam; anti–β-actin, anti-HA antibodies, anti-GST HRP conjugate antibody, HA–agarose beads, and Myc–agarose beads were purchased from Sigma-Aldrich.
Techniques: Activity Assay, Isolation, Staining, Confocal Microscopy, Immunoprecipitation, Purification, Incubation, Transfection, Luciferase, Plasmid Preparation, Expressing