Anti p105 p50

After 40h incubation with indicated stimuli, mDCs were washed with ice-cold PBS and lysed using NE-PER Nuclear and Cytoplasmic Extraction Reagents kit (Thermo Scientific, 78833). Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific, 78442) was used according to the manufacturer’s instructions. Protein concentrations of cytoplasmic and nuclear extracts were measured using Pierce BCA Protein Assay Kit (Thermo Scientific, 23227). Nuclear proteins of mDCs (25 μg/group) were pre-cleared by incubation with Protein A/G Magnetic Beads (Thermo Scientific, 88802) for 1h with continuous rotation at 4°C. After removal of beads, nuclear proteins were incubated with anti-p105/p50 (Cell Signaling Technology, 13586S) or anti-RelB (Abcam, ab33917) and Protein A/G Magnetic Beads overnight with continuous rotation at 4°C. Beads were then harvested, followed by extensive wash. Immunoprecipitants were eluted and denatured by heating with SDS-Sample Buffer (Boston BioProducts, BP-111R) at 90°C for 5 minutes. Proteins were then separated by electrophoresis in SDS/PAGE and transferred to PVDF membrane (Bio-Rad, 1620177) for immunoblotting.

Whole-cell proteins were extracted using RIPA lysis buffer supplemented with protease inhibitors (50 mM Tris–HCl, pH 7.4, 1% Nonidet P-40, 0.25% SDS, 150 mM NaCl, 1 mM EDTA, 1mM PMSF, 1mM NaF, 1mM Na₃VO₄, 2 µg/ml aprotinin, 1 µg/ml pepstatin, and 1 µg/ml leupeptin). Lysates were resolved on SDS-PAGE gels, transferred to nitrocellulose membranes and immunoblotted with anti-p105/p50 (Cell Signaling Technology), anti-BCL-3 (AbCam).