Mouse monoclonal anti flag antibody

An open reading frame (ORF) of OmPGRP-L1 was amplified from the OmPGRP-L1 full-length cDNA clone by PCR with a gene-specific primer set (PE-1F/PE-1R). The PCR fragment was digested with KpnI and XhoI (restriction sites underlined in the primers) and then subcloned into pcDNA3.1 (Invitrogen) digested with the same restriction enzymes. The resultant vector was named pcDNA3.1-OmPGRP-L1-FLAG. RTH-149 cells were transfected with pcDNA3.1-OmPGRP-L1-FLAG or an empty pcDNA3.1 vector using the Lipofectamine 2000 transfection reagent, according to the manufacturer’s instructions. OmPGRP-L1 overexpression was confirmed with western blotting using a mouse monoclonal anti-FLAG antibody (1:500, Cell Signaling Technologies, USA). At 48 h after transfection, cells (except control cells) were stimulated with 10 μg/ml iE-DAP and 100 μg/ml MDP for 24 h and collected for the analysis of target gene expression using qRT-PCR.