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Caspase-1 substrate (1.66 μl/ml 10 mM Ac-WEHD-AMC) was incubated with pancreatic tissue extracts with or without Caspase-1 inhibitor (Ac-WEHD-Al). For the alcohol and LPS experiments, AR42J cells were suspended and plated onto sterile coverslips (Nalgene, Thermo Scientific, Waltham, MA, USA) in six-well tissue culture plate at a density of 300 000 cells per well and incubated for 24 h. This data has be en c re ated by GSA Co ntent Generator DEMO! The calculated cycles were subtracted from the 18 s RNA (Applied Biosystems) reference, which was always included on the same plate. Five micrograms of total RNA was reverse-transcribed using SuperScript II (Life Technologies, Invitrogen) according to the manufacturer’s instructions. ibogaine uk was accomplished using rat-specific TLR-4 and MyD88 small interfering RNA (siRNA), control siRNA, and HyPerFect transfection reagent, according to the instructions (Qiagen). Twelve-thousand AR42J cells were grown on ibidi eight-well μ-slides for 24 h followed by RNA interference for TLR-4 and MyD88. Cells were suspended and plated on ibidi μ-slide (ibidi GmbH, Martinsried, Germany) at a density of 12 000 cells per well and incubated for 24 h. The supernatants were centrifuged (13 000 × g, 30 min, 4 °C) and the cytosolic fractions (supernatant) were collected. Protein concentrations in nuclear, cytosolic and mitochondrial fractions were determined by the BCA protein assay procedure (Pierce, Rockford, IL, USA). Neuronal cultures were treated with Aβ oligomers from CHO-conditioned medium, control vehicle from untransfected CHO cells, clustered recombinant EphrinB2-Fc (496-EB; R&D Systems, Minneapolis, MN, USA), or control Fc (SA1-600; Thermoscientific). They were exposed to starvation for another 24 h followed by incubating with either 10 μg/ml fluorescence-conjugated LPS-Alexa488 (Cy2) (Molecular Probes, Life Technologies, Thermo Scientific, Carlsbad, CA, USA) or unlabeled LPS, as well as 1.2 μg/ml per well recombinant CD14 and LBP (Abnova, Heidelberg, Germany), for an additional 16 h. Twenty-four hours after the transfection, the cells were exposed to LPS 10 μg/ml, as well as 1.2 μg/ml per well recombinant CD14 and LBP (Abnova), for an additional 16 h. Firstly, cells were pre-cultured in dissolution products of either TCP/HA or Sr-HT-Gahnite at 1/32 dilution for 7 days followed by seeding 4 × 104 HUVECs cells in DMEM containing 10% FBS in the upper chamber40. The pellets (heavy membranes enriched with mitochondria) were resuspended in lysis buffer containing 10 mM HEPES, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulphonyl fluoride, leupeptin, pepstatin and L-leucinethiol, each at 1 μg/ml, and 0.1% Nonidet P-40 pH 7.9, then, aliquoted and stored at −80 °C. 5) was fractionated according to a modified protocol, as previously described.52 Briefly, pancreatic frozen tissue was disrupted at 4 °C by a Teflon homogenizer in an extraction buffer containing 250 mM sucrose, 20 mM HEPES-KOH (pH 7.0), 10 mM KCl, 1 mM EGTA, 2 mM MgCl2, 1 mM EDTA, 1 mM DTT, 1 mM PMSF and protease inhibitor mixture (1 μg/ml each of pepstatin, leupeptin, and L-leucinethiol), and allowed to swell for 30 min at 4 °C.
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