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After determination of total protein by the Lowry assay, 10% poly

06/16/18 / Dr. Martin Buchwalter / Uncategorized

After determination of total protein by the Lowry assay, 10% polyacrylamide gels were equiloaded with samples, electrophoresed at 90 V, electrotransferred to PVDF membranes, and probed with primary mouse monoclonal antibodies for HMGB-1 or glutathione (Abcam, Cambridge, UK). Secondary goat antimouse horseradish peroxidase (HRP) was used (1:4,000). Blots were developed by ECL (Thermo Scientific, Asheville, NC). Total GSH from whole liver homogenates was measured using the Glutathione Assay Kit

(Cayman Chemicals, Ann Arbor, MI) according to the manufacturer’s protocol. For PCR assays, RNA was isolated from pancreas using a Qiagen RNEasy isolation kit (Qiagen, Germantown, MD). http://www.selleckchem.com/products/VX-809.html qPCR was performed using a standardized preconfigured PCR array (SA Biosciences, Frederick, MD) on the Stratagene MX3000P

(Promega, Madison, WI) according to the respective manufacturers’ protocols. (See Additional Supporting Methods.) To evaluate a possible role for DC in either exacerbating or protecting against APAP toxicity, we employed CD11c.DTR mice in which transient DC depletion can be effected (Supporting Fig. 1). Mice were depleted of DC or mock-depleted and then challenged with APAP. At 12 hours mice were sacrificed and the extent of liver injury determined by histopathology. Mice treated with APAP and depleted of DC (APAP-DC) had markedly more extensive INK 128 clinical trial centrilobular necrosis compared with controls (Fig. 1A,B). Consistent with these findings, serum liver enzymes were highly elevated in APAP-DC mice (Fig. 1C). Similarly, NPC production of inflammatory mediators after APAP challenge, including MCP-1 and IL-6, were higher in mice depleted of DC (Fig. 1D). DC depletion alone, in the absence of APAP challenge, had no effect (Fig. 1A-D). Similarly, effects of APAP were similar in both CD11c.DTR and WT mice, in the absence of Phosphoprotein phosphatase DC depletion (not shown). Notably, APAP metabolism appeared unchanged in APAP-DC mice compared with APAP treatment

alone based on tissue glutathione assay and glutathione adduct formation (Supporting Fig. 2A,B). To determine if there was higher systemic toxicity in APAP-challenged mice after DC depletion, we measured serum levels of inflammatory mediators. We found that serum MCP-1, IL-6, and TNF-α were elevated in APAP-DC mice (Fig. 1E,F). However, as expected, organ damage was limited to the liver, as the lungs, kidneys, pancreas, and intestine were histologically normal (Supporting Fig. 3). To determine whether DC depletion resulted in higher APAP-mediated mortality, mice were treated with APAP and depleted of DC or mock-depleted and then observed for up to 2 weeks. Remarkably, approximately half of APAP-DC mice died within 48 hours of challenge, whereas death was rare in control animals (Fig. 2). There was no further mortality observed in APAP-DC mice after 48 hours from the time of APAP challenge.

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