Anti phospho nf κb p65 ser536

The antibodies used in this study were as follows: anti-GST (Santacuze, #SC-138), anti-V5 (Invitrogen, #46–0705), or anti-IRF3 (Abcam, #ab25950), anti-phospho-IRF3 (Ser 396) (Cell signaling, #4947), anti-NF-κB p65 (Cell signaling, #4764), anti-phospho-NF-κB p65 (Ser536) (Cell signaling, #3031), anti-STAT1 (Cell signaling, #9175), anti-phospho-STAT1 (Cell signaling, #9167), anti-phospho-p38 (Cell signaling #9216), phospho-TBK1 (Cell signaling #5483), anti-NLRX1 (Proteintech, #17215-1-AP) and anti-His (Santacuze, #SC-1803) antibodies. The anti-FAF1 monoclonal antibody was provided by Dr. Eun-hee Kim (Department of Biology, Chungnam National University, Korea). The anti-interferon-α/β receptor (IFNAR) (25 μg/ml; Leinco Technologies) was pre-incubated in RAW264.7 cells and MEFs for 1 hr before VSV-GFP infection to block IFNAR.

Total protein was lysed in RIPA (Thermo Fisher Scientific) along with protease and phosphatase inhibitor cocktail, quantified by BCA Assay. Protein samples were separated on non-reducing SDS-PAGE 12% Tris–HCl gels (Beyotime, Shanghai, China) and then transferred onto PVDF membranes. The membrane was then blocked in 5% skimmed milk for 1 h, after which the membrane was incubated with primary antibodies diluted in primary antibody dilution buffer (Beyotime, Shanghai, China) overnight at 4 °C followed by incubation with horseradish peroxidase-conjugated secondary antibodies. Blots were developed with chemiluminescent detection reagent and imaged with a Chemiluminescent Imaging System (Tanon, Shanghai, China). Western blot quantification was conducted using ImageJ. The western blot antibodies, were listed as follows: anti-PRLR (Abcam, ab170935, 1: 1,000), anti-TNFSF13B (Abcam, ab224710, 1: 1,000), anti-GAPDH (Cell Signaling Technology, 5174, Massachusetts, USA, 1: 1,000), anti-NF-κB p65 (Cell Signaling Technology, 8242, 1: 1,000), anti-Phospho-NF-κB p65 (Ser536) (Cell Signaling Technology, 3033, 1: 1,000), anti-rabbit IgG (Cell Signaling Technology, 7074, 1: 5,000 in Tris Buffered Saline-Tween).

Cells were lysed in ice-cold lysis buffer with a protease inhibitor cocktail (Santa Cruz Biotechnology, Santa Cruz, CA) for 30 min and then centrifuged at 13,300 rpm for 20 min. Thirty micrograms of protein were separated on an SDS-PAGE gel and then transferred. After blocking, the membrane was incubated overnight at 4 °C with a primary antibody including anti-phospho-NF-κB p65 (Ser536), anti-NF-κB, anti-phospho-IκB-α (Ser32), anti-IκB-α, anti-phospho-IKKα/β (Ser176/180) (Cell Signaling Technology, Boston, MA), anti-phospho-IRE1α (Abcam, Cambridge, MA), anti-IRE1α (Abcam, Cambridge, MA), or anti-β-actin (Cell Signaling Technology) antibodies. After incubation with an HRP-conjugated secondary antibody, signals were detected with a chemiluminescence western blotting detection solution using Bio Imaging System (Syngene, Frederick, MD).

The RPMI 1640 medium, fetal bovine serum (FBS), and phosphate-buffered saline (PBS) were procured from Hyclone. Lipopolysaccharide (LPS, From E. coli, Cat no: L-2630) was procured from Sigma (MO, USA). Primers for real-time reverse transcriptase–polymerase chain reaction (RT-PCR) and TRIzol (Cat no: 15596026) were purchased from Invitrogen. Revert Aid First-Strand cDNA Synthesis Kit (Cat no: k1621) and FastStart Universal SYBR Green Master (ROX) (Cat no: 4385610) were purchased from Roche. RIPA (Cat no: 9806S) and primary antibodies for Western blot analysis, including anti-IκB (Cat no: 76041S), anti-phospho-IκB (Ser32) (Cat no: 2859S), anti-NF-κB p65(Cat no: 8242S), anti- phospho-NF-κB p65(Ser536) (Cat no: 3033) anti-JAK2(Cat no: 3230S), anti-phospho-JAK2(Tyr1007/1008) (Cat no: 3771S), and anti-GAPDH (Cat no: 5174S), were purchased from Cell Signaling Technology. Secondary antibodies were obtained from Sigma. The Enhanced Chemiluminescence Solution Kit (Cat no: WBKLS0500) was obtained from Millipore (MA, USA). APC mouse anti-human CD16(Cat no: ab203883) and FITC mouse anti-human CD86(Cat no: ab213044) were purchased from Abcam (Cambs, UK). APC mouse anti-human CD40(Cat no: 555591) and FITC mouse anti-human CD23(Cat no: 561146) were purchased from BD Systems (BD Biosciences, USA).

To measure the levels of HBsAg and HBeAg, culture supernatants of transfected cells were diluted 5-fold and analyzed by ELISA as described previously [35 (link)]. Levels of HBsAg, HBeAg and antibody to HBsAg in mouse sera were measured by ELISA after a 1:10 dilution [35 (link)]. Western blot analysis was performed as previously described [35 (link)]. The following antibodies were used: rabbit polyclonal anti-HBc and mouse monoclonal antibodies (mAbs) against β-actin from Santa Cruz Biotechnology, anti-GAPDH (Proteintech), anti-FLAG-tag and rabbit monoclonal Abs against HA-tag from Sigma-Aldrich, anti-phospho-IRF3 (Ser396), anti-IRF3, anti-phospho-IκB (Ser32), anti-IκB, anti-phospho-NF-κB p65 (Ser536) and anti-NF-κB p65 from Cell Signaling Technology. Proteins were visualized with appropriate HRP-conjugated secondary antibodies (Jackson Immuno Research) and SuperSignal-Femto chemiluminescent substrate (Pierce).