Lipofectamine ltx plus reagent

To detect the activation of the NF-κB pathway induced by BVDV infection, BT cells (over 80% confluent in 24-well plates) were transfected with the pNF-κB-luc reporter plasmid (0.44 μg/well) and internal reference plasmid pRL-TK (0.06 μg/well) using lipofectamine® LTX & Plus Reagent (Invitrogen, USA). At 12 hours after transfection, the transfected BT cells were infected with cpBVDV AV69 at a multiple of infection (MOI) of 1.0, using the UV-inactivated BVDV as control. The cell samples were collected at 12 h, 24 h, and 48 h after BVDV infection, followed by the determination of the firefly luciferase and the Renilla luciferase activities using a Dual-Luciferase® Reporter Assay System (Promega, USA). To screen the key viral proteins and the key functional domains involved in activating the NF-κB signalling pathway, BT cells or 293T cells (over 80% confluent in 24-well plates) were co-transfected with recombinant eukaryotic plasmids expressing BVDV proteins (or truncated proteins), pNF-κB-luc, and pRL-TK, using lipofectamine® LTX & Plus Reagent (Invitrogen, USA), followed by the determination of the NF-κB relative dual-luciferase activities. In parallel, co-transfection with empty plasmid pCMV-HA was used as negative control. All reporter gene assays were performed in triplicates and repeated thrice. Data are presented as the mean ± SD values.

Human embryonic kidney epithelial 293T cells were cultured in Dulbecco’s modified Eagle’s medium with high glucose and L-glutamine (DMEM) (Invitrogen, Canada) supplemented with 10% fetal bovine serum (FBS, Invitrogen). MDA-MB-231 human breast cancer cells, purchased from American Type Culture Collection (ATCC, Manassas, VA, USA), were maintained in 10% FBS-supplemented DMEM. 293T cells were transiently transfected using the calcium phosphate precipitation method, and MDA-MB-231 cells were transfected using Lipofectamine LTX Plus reagents (Invitrogen, Canada) [12 (link),16 (link)]. MDA-MB-231 cells were transfected with pCaGip vector or pCaGip plasmid encoding SnoN (WT), SnoN (KdR) or SUMO-SnoN using Lipofectamine LTX Plus reagents and incubated with1 μg/ml puromycin (Invitrogen, Canada) containing complete medium [11 (link)].

HCASMCs were transfected in 12‐well dishes with 0.2 µg of WT or SNP primary miR143/145 psiCheck2 reporter. Transfection was performed with Lipofectamine LTX+Plus reagent (Life Technologies), following the manufacturer's protocol. Finally, cells were harvested 48‐h post‐transfection and analysed using the dual‐luciferase reporter assay system (Promega) as described by the manufacturer.