RANKL signaling molecules in MRL/lpr DCs. (A) The expressions of TRAF6, IKKα, and phospho-IκB (pIκB) of RANKL-stimulated BMDCs from C57BL/6, MRL^+/+, and MRL/lpr mice were detected using the cytoplasmic extracts by Western blot analysis. The BMDCs were stimulated with RANKL (100 ng/mL) from 0 to 120 minutes. GAPDH was used as a housekeeping protein. (B) Nuclear translocation of NF-κB (p65 and p50) of RANKL-stimulated BMDCs from C57BL/6, MRL^+/+, and MRL/lpr mice was detected by immunoblot. Histone was used as a housekeeping protein of the nuclear extracts. (C) Increased TRAF6 and p-IκB in the cytoplasm, and accelerated nuclear translocation of p50 and p65 in the nucleus of RANKL-stimulated MRL/lpr DCs were detected by confocal microscopic analysis. BMDCs from MRL/lpr and MRL^+/+ mice were stimulated for 48 hours with or without RANKL, fixed in 3% PFA on a glass slide, and stained with anti-p65, anti-p50, TRAF6, and p-IκBα followed by Alexa Fluor 488–labeled (green) or Alexa Fluor 568–labeled (red) anti-mouse or anti-rabbit IgG as the second antibodies. The nuclei were stained with DAPI. Original magnification, × 630. All data are representative of 3 to 5 independent experiments.