Anti p65 c 20

pCMV/Myc-TRAF6, TRAF2, and TRAF5 was provided by Dr. Lingqiang Zhang (State Key Laboratory of Proteomics, Beijing, China). pEFNeo/HA-CHIP, pRKIM/Myc-CHIP, and pRKIM/Myc-CHIP (H260Q) were generated in our lab as described previously.^{23 (link)} I-κBα, phosphor-I-κBα (Ser32) and anti-α-tubulin (3873p) were purchased from Cell Signaling Technology (Beverly, MA). Anti-TRAF6 (H-274) and anti-p65 (C-20) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-CHIP antibody was developed in our lab.^{16 (link)} MG132 was purchased from Millipore (Billerica, MA). Cyclohexanone (CHX) was purchased from Amresco (AMRESCO Inc., OH).

Cell cytoplasmic and nuclear extracts were obtained by using NE-PER nuclear and cytoplasmic extraction reagents according to the manufacturer’s protocols (ThermoFisher). Immunoblotting was performed as previously described (8 (link)). The blots were probed with anti-IRF7 antibody (EPR4718; abcam), anti-Lamin-B (M-20; Santa Cruz Biotechnology), anti-IKKα (Cell Signaling), anti-AKT (Cell Signaling), anti-Osteopontin (Abcam), anti-p65 (C-20; Santa Cruz Biotechnology), anti-P50 (Santa Cruz Biotechnology), anti-STAT1 (Cell Signaling), anti-IRAK-M (ProSci), and anti-Blimp-1 (Abcam). The activation of IRF7, IKKα/β, AKT, and STAT1 were detected by phospho-specific antibodies against pIRF7 (Ser471/472; D6M2I; Cell Signaling), pIKKα/β (Ser176/180; 16A6; Cell Signaling), pAKT (Ser473; D9E; Cell Signaling), and pSTAT1 (Tyr701; 58D6; Cell Signaling). Representative blots from at least two independent experiments were shown.
Rac1 activation was detected by Rac1 activation assay kit (Abcam). Briefly, the total cell lysates were harvested from stimulated FLpDCs and incubated with PAK1 PBD beads at 4°C for 1 h. Rac1-GTP precipitate and the total lysate controls were analyzed by western blot analysis. Rac1 was detected by a specific mouse monoclonal antibody.