The cell lysates were collected for luminescence quantification using the protocol DLR-0-INJ (with 10 s integral time) of the GloMaxTM Luminometer (Promega). Idasanutlin manufacturer Ten microliter of each sample was treated with 50 μL of Luciferase Assay Reagent II to obtain the first measurement, while the second measurement was acquired upon addition of 50 μL Stop & Glo® Reagent. The ratio of the first and second luminescence readings was taken as the
level of desired plasmid activation. The Stealth siRNA (Invitrogen) designated S1, S2 or S3 were designed to target human SARM in three different domains. HEK293 cells were seeded into 24-well plates at a density of 1×105 cells/well in 0.5 mL medium, and were transfected with expression vectors and luciferase reporter genes together with siRNA for 24 h. Then the cells were harvested and divided into two halves, one for measurement of SARM mRNA level by end-point PCR and the other for luciferase assay. To examine the effect of LPS stimulation on SARM mRNA expression, HEK-293 or U937 cells were seeded into 6-well plates at a density of 2.5×105 cells/well in 2 mL medium. One day after transfection with the relevant plasmids, the cells were stimulated with 10 ng/mL LPS for another 24 h, and the reporter gene assay was performed. The IL-8 was measured with OptEIA™
(BD, San Jose, CA, USA) according to the manufacturer’s instructions. The wells were coated with 100 μL capture antibody selleck kinase inhibitor diluted in
coating buffer. The plate was sealed and incubated overnight at 4°C. After three washes, the wells were blocked with 200 μL assay diluents at room temperature for 1 h, followed by another three washes. Then, 100 μL diluted IL-8 standard and test samples were added and incubated for 2 h at room temperature. After repeated washes, the substrate was added and incubated for 20 min at room temperature, and the OD405nm was read. Total RNA from cells was isolated with TriZol Reagent (Invitrogen) and reverse-transcribed with SuperScript II reverse transcriptase (Invitrogen) using Oligo(dT) as primer. The resulting cDNA was used to determine the relative amount of SARM mRNA either by end-point PCR with Taq DNA polymerase (Invitrogen), or by real-time PCR with SYBRGreen (ABI) using the ABI Prism SDS 7000 sequence detection system. β-Actin Branched chain aminotransferase was used as internal control in both cases. In total 2.5×106 HEK-293 or U937 cells were seeded in 60-mm dishes. HEK-293 cells were transfected for 24 h with TRIF- or MyD88-expressing plasmid, along with a plasmid expressing SARM. U937 cells were treated with 10 ng/mL LPS. Cells were lysed in Laemlli sample buffer, and lysates were resolved in 12% SDS-PAGE gel and electroblotted (Biorad). The PVDF membrane was blocked with 5% skimmed milk in PBST (PBS containing 0.05% v/v of Tween-20) for 1 h and washed three times with PBST, followed by incubation overnight at 4°C with primary antibody.