Anti phospho nf κb p65 ser536 antibody

Compound Vam3, synthesized from resveratrol, was provided by the Institute of Materia Medica, Chinese Academy of Medical Sciences (Beijing, China; Huang et al., 1999 ). The purity of Vam3 was above 98% as determined by ¹H-NMR spectra. Rat basophilic leukemia cell lines (RBL-2H3) and murine macrophage cell lines (RAW264.7) were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). LPS (serotype O127:B8), AOM, disodium cromoglycate (DSCG), dinitrophenol (DNP)-specific IgE, and DNP-conjugated bovine serum albumin (DNP-BSA) were purchased from Sigma–Aldrich (St. Louis, MO, USA). DSS (MW = 36,000–50,000) was purchased from MP Biomedicals, LLC (Solon, OH, USA). FITC-conjugated anti-mouse CD45 antibodies, PE.Cy7-conjugated anti-mouse CD11b antibodies, and APC-conjugated anti-mouse Gr1 antibodies were purchased from eBioscience (San Diego, CA, USA). Recombinant mMCP-1 was purchased from Sino Biological Inc. (Beijing, China). Anti-CD117 antibody was purchased from Dako (Carpinteria, CA, USA). FuGENE HD Transfection Reagent was purchased from Promega (Madison, WI, USA). Anti-phospho-NF-κB p65 (Ser536) antibodies, anti-NF-κB p65 antibodies, and anti-β-actin antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA).

Equal amounts of total protein (50 µg) from each lysate were subjected to 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% skim milk in Tris-buffered saline containing Tween 20 and incubated overnight with anti-phospho-NF-κBp65 (Ser536) antibodies (1:1000; Cell Signaling Technology, Danvers, MA, USA) and anti-NF-κBp65 antibodies (1:1000; Abcam, Cambridge, UK) at 4 °C. The membranes were washed thrice with wash buffer (Beyotime Biotechnology, Jiangsu, China) and incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies (1:1000; Beyotime Biotechnology, China) for 1 h at room temperature. Lastly, the membranes were subjected to DAB staining to detect the protein bands.

NT2 aggregates and N9 protein samples western blot analysis was performed accordingly to^{41 (link)}. Aggregates were lysed with TX-100 lysis buffer (50 mM Tris, 5 mM EDTA, 150 mM NaCl, 1% Triton X-100, pH 7.4) and N9 protein extraction was performed with RIPA buffer. Primary antibodies were incubated overnight at RT, followed by secondary antibodies (horseradish peroxidase-conjugated, ECL anti-mouse IgG, anti-rat IgG or anti-rabbit IgG; Pierce, Millipore and GE Healthcare), incubated for 2 h at room temperature. Anti-GAPDH antibody (Thermo Scientific) was used as loading control. Primary antibodies used for protein detection were: anti-βIII-Tubulin (Millipore), anti-GFAP (DAKO), and MitoProfile Total OXPHOS WB primary antibody cocktail (Abcam), anti-phospho-NF-κB p65 (ser536) antibody (Cell Signalling), anti-NF-κB p65 (C-20) (Santa Cruz Biotechnology), and anti-IκB-α (C-21) antibody (Santa Cruz Biotechnology). Membranes were developed using Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare) and visualized using a ChemiDocTM XRS + System (BioRad).