Tlrl picw

The synthetic lipopeptides Pam₃Cys-Ser-Lys₄ (P₃C) and fibroblast-stimulating lipopeptide 1 (FSL-1) were from EMC (Tübingen, Germany). LPS from Escherichia coli O111:B4, MDP, and poly(I:C) were purchased from Invivogen (San Diego, CA). Vaccigrade poly(I:C) (Invivogen, vac-pic) was used in most experiments. HMW (Invivogen, tlrl-pic) and LMW poly(I:C) (Invivogen, tlrl-picw) were used for comparison of high- and low-molecular-weight poly(I:C). Recombinant TNF was purchased from Peprotech (Rocky Hill, NJ).

To evaluate the effect of Poly (I:C) on focal cerebral I/R injury, we employed a non-preconditioning regimen. Poly (I:C) (Catalog Cod: tlrl-picw, InvivoGen, San Diego, CA, USA) was dissolved in sterile endotoxin-free 0.9% NaCl and injected intraperitoneally (i.p., 10 μg/25 g bodyweight, n + 8) 1 hr prior to cerebral ischaemia (60 min.) followed by reperfusion for 24 hrs.
To investigate the therapeutic effect of Poly (I:C) on focal cerebral I/R injury, Poly (I:C) (i.p., 10 μg/25 g bodyweight, n + 8) was administered by intravascular injection 30 min. after the beginning of cerebral ischaemia. Focal cerebral ischaemia was continued for an additional 30 min. followed by reperfusion for 24 hrs.
To examine the role of TLR3 in Poly (I:C)-induced protection against cerebral I/R injury, TLR3 KO mice (n + 7/group) were treated with or without Poly (I:C) (10 μg/25 g bodyweight) 1 hr before the mice were subjected to focal cerebral ischaemia (60 min.) followed by reperfusion (24 hrs). The infarct size for all experiments was determined by triphenyltetrazolium chloride (TTC) staining as described below 9 (link),10 (link),13 (link),25 (link),26 (link).

HEK293T cells were cotransfected with the IFN-β promoter–firefly luciferase (FLuc) reporter plasmid (pIFN-β-FLuc), p3×Flag-hHB, and the internal reference reporter TK-Renilla luciferase (RLuc) as an internal control (pRLuc-TK). The total amounts of the plasmid DNAs were equalized with the empty control vector p3×Flag-CMV-10 (p3×Flag-EV). At 24 hpt, the cells were infected with SeV or phosphate-buffered saline (PBS) for another 24 h. Then cells were lysed, and the activities of the reporter genes were determined using a dual-luciferase reporter assay system (10 pack; Promega). The luciferase induction mediated by IFN-β promoter (IFN-β-Luc) was presented as relative expression level of FLuc/RLuc. For the RIG-I- or MDA5-mediated response, HEK293T cells were cotransfected with pIFN-β-FLuc and pRLuc-TK as well as with pMyc-RIG-I, pMyc-MDA5, short poly(I·C) (catalog no. tlrl-picw; InvivoGen), or long poly(I·C) (catalog no. tlrl-pic; InvivoGen). The luciferase activities were measured at 24 hpt, and relative expression levels were calculated as described above.

A549, A375, BSC40, and MDCK cells were cultured in Dulbecco´s modified Eagle´s medium supplemented with 10% fetal bovine serum (FBS), 1% L-Glutamine (G7513, Sigma-Aldrich), and 1% Penicillin-Streptomycin (P4333, Sigma-Aldrich). Vero, Vero-E6, and HeLa cells were grown in Minimal Essential medium supplemented with 10% FBS, 1% L-Glutamine, and 1% Penicillin-Streptomycin. Treatment with the spermidine analogue and competitive Deoxyhypusine Synthase (DHS) inhibitor N1-guanyl-1,7-diamine-heptane (GC7) (259545, Millipore) was carried out at a concentration of 20 µM unless otherwise indicated. IKK 16 (Abcam) and BAY 11-7082 (Sigma-Aldrich) were used at a concentration of 100 nM and 1 μM, respectively. Poly I:C (tlrl-picw, In vivogen) was used at a final concentration of 5 µg/ml. Smart-pool small interfering RNAs (siRNAs) against eIF5A1 (77LQ-HUMAN_XX-0020, sieIF5A) and on-target plus non-targeting siRNAs (77D-001810-10-50, siC) were purchased from Dharmacon. A retrovirus vector expressing short hairpin RNA (shRNA) targeting p65 subunit of NF-kB was kindly provided by Scott Lowe lab (Chien et al., 2011 (link)) (Memorial Sloan Kettering Cancer Center, NY; USA).

PAR from a PARP1 autoPARylation reaction was isolated, fluorescently labeled, and analyzed with PAGE essentially as described (98 (link)). AutoPARylated mCherry-PARP1 was acid precipitated, and PAR was isolated with base and ethanol precipitation as described above for PAR16 synthesis. The mixture of PAR lengths (~250 μM ADPr) was labeled with Cy3-dATP (10 μM, Jena Bioscience #NU-835-CY3), poly(I:C) RNA (50 μg/mL, Invivogen #tlrl-picw), and OAS1 (1 μM) in 25 mM HEPES pH 7.5, 20 mM MgCl₂, 2.5 mM DTT at 37 °C for 2 hrs. Labeling reactions were diluted with Formamide-EDTA loading buffer and separated with 12% urea-PAGE (National Diagnostics # EC-833) in an adjustable slab gel apparatus (VWR #CBASG-400) equipped with 28 cm plates. Cy3 signal was measured with a Licor-M. The image was exported and annotated with ImageStudio and Adobe Illustrator.