Donkey anti goat igg sc 2020

MDCK cells were seeded on a 24-well plate at a density of 1 × 10⁵ cells/well. 2–5 (12.5 μM), 1 (12.5 μM, positive control), or DMSO (0.125%, negative control) were mixed with MOI 0.1 of A/PR/8/34 virus and incubated for 30 min prior to the cells being added. At 4, 8, 12, or 24 h post-infection, the cells were lysed in a buffer containing 125 mM Tris-HCl, pH 6.8, 5% sodium dodecyl sulfate, 25% glycerol, 0.1% bromophenol blue, and 10% β-mercaptoethanol and boiled for 5 min. The cell lysates were separated on a 10% polyacrylamide gel. The proteins were transferred to a polyvinylidene fluoride microporous membrane (Millipore, MA, USA). FluA-NP 4F1 (SouthernBiotech) or a goat anti-influenza A viral NS1 antibody (vC-20; Santa Cruz Biotechnology, CA, USA) were used as primary antibodies to detect their respective proteins. A rabbit anti-β-ACTIN antibody (13E5; Cell Signaling, MA, USA) was used as an internal control. The secondary antibodies, horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (SouthernBiotech) or donkey anti-goat IgG (sc-2020; Santa Cruz Biotechnology), were used as appropriate. The signals were detected using Immobilon Western Chemiluminescent HRP Substrate (Millipore). Signal intensities were measured using ImageJ software, and the protein levels of NP and NS1 were normalized to that of β-actin.

P6 was synthesized according to Di Nunno et al. (15 (link)), whereas mofezolac was synthesized following Micetich’s protocol (16 ). All the other reagents and solvents were purchased from Sigma-Aldrich (Milan, Italy) and used without any further purification.
Lipopolysaccharide from Escherichia coli serotype 0127:B8 was purchased from Sigma-Aldrich (Milan, Italy). The goat polyclonal a p-IκB (sc-7977) antibody (Ab) was purchased from Santa Cruz Biotechnology (DBA, Milan, Italy); COX-1 (Ab 695) and COX-2 (Ab 15191) Abs were obtained from Abcam (Cambridge, UK). Goat anti-rabbit IgG (sc-2004), goat anti-mouse IgG (sc-2005), and donkey anti-goat IgG (sc-2020) were purchased from Santa Cruz Biotechnology; mouse primary monoclonal antibody (mAb) anti-glial fibrillary acidic protein (GFAP) (Merck Millipore, Milan, Italy), mouse mAb anti-ionized calcium-binding adapter molecule-1 (Iba-1) (Merck Millipore). Elisa kit for PGE₂ evaluation was purchased from Cayman Chemical (Ann Arbor, MI, USA). MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], 3,3′-diaminobenzidine, and tribromoethanol were obtained from Sigma-Aldrich, Milan, Italy.

The cells were lysed in a buffer containing 125 mm Tris-HCl, pH 6.8, 5% SDS, 25% glycerol, 0.1% bromphenol blue, and 10% β-mercaptoethanol and boiled for 5 min. The cell lysates were then separated on a 10% polyacrylamide gel. The proteins were transferred to a polyvinylidene fluoride microporous membrane (Millipore). FluA-NP 4F1 (SouthernBiotech), a goat anti-influenza A viral NS1 antibody (vC-20, Santa Cruz Biotechnology, Inc.), a rabbit anti-firefly luciferase polyclonal antibody (MBL, Nagoya, Japan), and a rabbit anti-Renilla luciferase polyclonal antibody (MBL) were used as primary antibodies to detect their respective proteins. A rabbit anti-β-actin antibody (13E5, Cell Signaling) was used as an internal control. The secondary antibodies, horseradish peroxidase-conjugated goat anti-mouse IgG (SouthernBiotech), donkey anti-goat IgG (sc-2020, Santa Cruz Biotechnology), or goat anti-rabbit IgG (KPL), were used as appropriate. The signals were detected using Western Lightning ECL Pro (PerkinElmer Life Sciences). Signal intensities were measured using ImageJ software, and the protein levels of firefly and Renilla luciferase were normalized to that of β-actin.