Ecl enhanced chemiluminescence detection kit

Agroinfiltrated N. benthamiana leaves were harvested and homogenized in liquid nitrogen, followed by addition of equal volumes of gel loading buffer (100 mM Tris-HCl, pH 6.8, 20% glycerol, 4% SDS, 200 mM β-mercaptoethanol, 0.2% bromophenol blue). After vortex mixing and boiling, the samples were centrifuged for 10 min at 12000 g. Proteins remaining in the supernatant were resolved by 12.5% SDS-PAGE, followed by staining with Coomassie brilliant blue or were transferred to nitrocellulose filters for Western blot analysis [85 (link)].
Western blots were performed as described previously [67 (link), 84 (link)]. Briefly, nitrocellulose membranes containing transferred proteins (Hybond-C, GE Healthcare) were blocked, incubated with primary antibodies raised against the γb, Actin, GFP, FLAG or HIS proteins. After washing, the membranes were incubated with secondary antibodies conjugated to horseradish peroxidase or protein A-alkaline phosphatase (Sigma-Aldrich), and the signals were detected with an enhanced chemiluminescence (ECL) detection kit (GE Healthcare) or by color reactions developed by incubating with substrate solution (0.33 mg/mL NBT and 0.165 mg/mL BCIP in 100 mM Tris-HCl buffer, pH 9.5, containing 100 mM NaCl). The results were recorded with a Cannon scanner.

Cell lysates were prepared by scraping the cells with Laemmli Sample Buffer (LSB) containing 4% Sodium Dodecyl Sulfate SDS, 20% Glycerol, 10% β-Mercaptoethanol, 0.004% Bromophenol Blue, and 0.125 M Tris-HCl (pH = 6.8). The resulting lysates were boiled for 5 min. Protein samples were separated by SDS-PAGE gels and transferred to PVDF membranes. Membranes were then blocked either by 5% BSA for 2 h at room temperature depending on the primary antibody used. The membranes were then incubated with the appropriate primary antibody for 2 h. After that, the membranes were washed and incubated with secondary antibodies at a concentration of 1:2000 for 1 h at room temperature. Bands were visualized by treating the membranes with a chemiluminescent reagent Enhanced Chemiluminescence-ECL detection kit (GE Healthcare, Chicago, IL, USA). The levels of expression of proteins were compared by densitometry using the ImageJ software and plotted in Excel.

Cell culture medium and all the other materials required for cell culture were purchased from Gibco BRL Life Technologies (Grand Island, NY, USA). LPS (Escherichia coli O55:B5), dimethyl sulfoxide (DMSO), bovine serum albumin (BSA), and the specific protein kinase inhibitors (PD98059 and SP600125) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). CellTiter⁹⁶ AQ_ueous One Solution Cell Proliferation assay kit, dual-luciferase assay system, murine NF-κB promoter/luciferase DNA, pRL-TK DNA, and moloney murine leukemia virus (M-MLV) reverse transcriptase were obtained from Promega (Madison, WI, USA). Enzyme-linked immunosorbent assay (ELISA) kits for TNF-α, IL-1β, and IL-6 were obtained from eBioscience (San Diego, CA, USA) and PGE₂ ELISA kit was purchased from R&D Systems (Minneapolis, MN, USA). Primary and secondary antibodies were purchased from Cell Signaling Biotechnology (Danvers, MA, USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA), respectively. 4’,6-Diamidino-2-phenylindole (DAPI), Lipofectamine Plus Reagent, TRIzol, and Alexa Fluor® 488-conjugated secondary antibody were purchased from Invitrogen (Carlsbad, CA, USA). The enhanced chemiluminescence (ECL) detection kit was purchased from GE Healthcare Life Sciences (Piscataway, NJ, USA).

The reverse line blot assay was carried out according to Kong et al.[6 (link)]. Briefly, a Biodyne C membrane was activated with 16% (w/v) 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) for 20 min and rinsed with sterile milliQ water. The membrane was placed in a Miniblotter® (Immunetics) and specific 5’ amine labeled oligonucleotide probes were then bound to the membrane and fixed with 0.1 M NaOH. The membrane was then removed, rotated 90° and placed back into the Miniblotter. Products from the five mPCR were combined and boiled for 5 min before being applied to the membrane perpendicular to the bound probes. The membrane was then incubated at 55°C for 1 hr before washing in 1× SSPE (150 mM sodium chloride, 10 mM sodium phosphate and 1 mM EDTA)/0.1% SDS solution. A streptavidin-peroxidase conjugate (Roche) was then added and the membrane further incubated at 42°C for 1 hr before washing and exposing with enhanced chemiluminescence (ECL) detection kit (GE LifeSciences). Chemiluminescence was detected using LAS3000 Imager (Fujifilm).
A clear hybridisation signal was interpreted as the presence of the corresponding gene, whereas the lack of signal was interpreted as the absence of a gene. Faint or indistinct hybridisations deemed ambiguous were confirmed using individual PCR and agarose gel electrophoresis.