Ac yvad cho

Cells were seeded at 1.4×10⁴ cells/well onto 384-well cell culture plates in RPMI1640 medium containing 10% FBS and compounds. Four hours later, the cells were stimulated with 20 ng/ml LPS for 18 hours. After centrifugation, the supernatants were transferred to 384-well small-volume white plates using a Biomek NX^P (Beckman Coulter). The relative cytokine levels were measured using an HTRF Kit (Cisbio Bioassays, France) and a POWERSCAN4 Microplate Reader (DS Pharma Biomedical, Japan) with excitation at 330 nm and emission at 620 and 665 nm. The HTRF signals were calculated as the HTRF ratio (10000×Em 665 nm/Em 620 nm), and then the percent inhibition was calculated using DMSO controls. Cell seeding and reagent dispensation were performed with a Multidrop Combi (Thermo Fisher Scientific). The reagents purchased are as follows: Ac-YVAD-CHO, MG-132 (Merck KGaA), MCC950 (Sigma-Aldrich), FDA-approved drug library, ICCB Known Bioactives Library (Enzo Life Sciences, USA), US-Drug Collection, International Drug Collection (MicroSource Discovery Systems, USA), LOPAC1280 (Sigma-Aldrich), Tocriscreen mini (Tocris Bioscience, UK), and Kinase Inhibitor Libraries (Enzo Life Sciences, Merck KGaA, Selleck Chemicals (USA)).

RPMI 1640 medium and DMEM, fetal bovine serum (FBS), glutamine, and a streptomycin/penicillin antibiotic solution were all purchased from Invitrogen. Bacterial lipopolysaccharide (LPS, Escherichia coli 026:B6) and staurosporine (STS)² were purchased from Sigma. Ac-YVAD-CHO, IETD-CHO, cycloheximide (CHX), ALLN, calpain inhibitor III, EST, and PD150606 were purchased from Merck Chemicals, Ltd. CHX causes apoptosis by inhibiting protein translation and subsequently cell growth, whereas STS is a broad spectrum kinase inhibitor that induces apoptosis. Z-VAD-fmk was purchased from Promega. Necrostatin-1 was purchased from Sigma. The anti-mouse IL-1β and IL-1α antibodies were from R&D Systems. Secondary antibody HRP conjugates were from DAKO.

Human NGF-β (Calbiochem, San Diego, CA, USA), K252a (Axxora LLC, Farmingdale, NY, USA), and PLX7486 (TrkA inhibitor, kind gift from Plexxikon, Berkeley, CA, USA) were used at 100 ng/ml, 100 ng/ml and 1 μM respectively. LPS (Sigma, St. Louis, MO, USA), Ac-YVAD-CHO, Caspase-1 inhibitor [38 (link)] (YVAD, EMD Millipore, Billerica, MA,USA), and pyrrolidine-dithio-carbamate (PDTC), NF-κB inhibitor (Sigma) [39 (link)] were used at 1 μg/ml, 50μM, and 50μM respectively. Anti-p75-NTR rabbit anti-mouse polyclonal antibody (EMD Millipore, AB1554) was used in 1:1000 dilution (1μg/ml as per manufacturer’s instruction) for blocking p75-NTR as per manufacturer’s instructions. The p75-NTR blocking antibody is a transmembrane glycoprotein consisting of an extracellular domain responsible for ligand binding.