Anti lamin b m 20

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.

Cells were harvested at indicated time points and lysed in RIPA buffer (Sigma). Western blot analysis was performed with the use of conventional protocols as described previously [22 (link)]. Nuclear and cytoplasmic proteins were extracted in accordance with the manufacturer's instructions (Pierce Biotechnology). The antibodies and dilutions used included anti-β-actin (AC-15; 1:2000; Sigma), anti-Keap1 (D1G10; 1:1000; Cell Signaling Technology), anti-Nrf2 (D1Z9C; 1:1000; Cell Signaling Technology), anti-LaminB (M-20; 1:1000; Santa Cruz), anti-GFP (A00185.01; 1:1000; Santa Cruz). After extensively washed, the membranes were incubated with anti-mouse or anti-rabbit IgG-horseradish peroxidase conjugate antibody (Zhongshan Company) for 1 h at room temperature and developed with a Luminol chemiluminescence detection kit (Santa Cruz). Membranes were reprobed for β-actin antibodies for normalization and accurate quantification. Protein expression level was quantified by using a Gel EDAS 293 analysis system (Cold Spring USA Corporation) and Gel-Pro Analyzer 3.1 software (Media Cybernetics).