X tremegene hp dna transfection reagent

tsBN2,⁴⁰ (link) Baby hamster kidney 21 cells (BHK-21) or HeLa were grown and maintained in 1× Dulbecco's modified Eagle's (DMEM) F12 medium (ThermoFischer Scientific, #11330–032) or DMEM glutamax (ThermoFischer Scientific, #21885–025) supplemented with 10% fetal bovine serum (FBS, v/v) (ThermoFischer Scientific, #10500064) and 1% penicillin-streptomycin (v/v) in a humidified atmosphere containing 5% CO2. The BHK-21 and HeLa cells were maintained at 37°C, whereas tsBN2 cells were cultured at 33°C. For interaction studies, the cells were treated for 4 h at 33°C (permissive temperature) or 39.5°C (restrictive temperature) before crosslinking. For transfection of cells with plasmids encoding Samp1-YFP, X-treme gene HP DNA transfection reagent (Sigma, #000000006366236001) was used and cells were analyzed 24 h post-transfection.

A549 cells were respectively cotransfected with pcDNA3-PB1, pcDNA3-PB2, pcDNA3-PA, or pcDNA3-NP, and pol I-WSN-NS non-coding region minigenome reporter, using X-tremeGENE HP DNA Transfection Reagent (Sigma), as well as 20 nM of VIM siRNA (4390824, Silencer Select siRNAs; Thermo Fisher Scientific) or control siRNA (4390843, Silencer Negative Control No. 1 siRNA; Thermo Fisher Scientific), using Lipofectamine RNAiMAX (Invitrogen) transfection reagent according to the manufacturer’s instructions. At 48 h after transfection, the cells were harvested and fractionated according to the experimental procedures described in the handbook provided along with the NE-PER Nuclear and Cytoplasmic Extraction Reagents (78833; Thermo Fisher Scientific). Approximately 60 μg of protein lysate from the cytosol fraction and lysate from the nuclear fraction at half the volume of the cytosol fraction were further used to analyze the expression of NP, vimentin, LaminB1, and GAPDH, using western blotting.

HEK293 cells (American Type Culture Collection) were maintained in DMEM base media supplemented with 10 % FBS (Gemini Bio-Products, NC), 100 μg/ml streptomycin, and 100 U/ml penicillin (Gibco). Cells were cultured at 37 °C and 5 % CO₂ and were sub-cultured upon reaching 80 % confluence. For transfection, 2.5 × 10⁵ cells were seeded in 12-well plates in triplicates per each condition. The following day, cells were transfected for 24 h with X-tremeGENE™ HP DNA Transfection Reagent (6,366,236,001, Sigma-Aldrich) and 1 μg/well plasmid DNA constructs expressing pcDNA3.1/NT-GFP-TOPO™ (K481001, NT-GFP Fusion TOPO™ Expression Kit, Invitrogen™), CS2 Flag-SMAD3 (14052, Addgene), and mutant CS2 Flag-SMAD3 (p.I67S). After transfection, cells were stimulated with 5 ng/mL of TGFβ-1 (7754-BH, R&D) for another 24 h and harvested for RNA.

Variable light-chain (VL) and variable heavy-chain (VH) domains of the anti-CD3 OKT 3 hybridoma and the anti-CD16 3G8 hybridoma were cloned into a pcDNA 3.1 vector (Invitrogen, Carlsbad, CA) by standard cloning techniques, followed by the DNA sequence of the LRPAP1 epitope, respectively (Supplemental Digital Figure 1, http://links.lww.com/HS/A179). The single-chain fragment (scFv) containing the variable heavy and light chain of the anti-CD16 hybridoma, linked by a glycine-serine linker, was bought from GenScript (GenScript USA Inc., Piscataway, NJ).
The primers used for PCR amplification of anti-CD3 scFv and the LRPAP1 epitope are shown in Table 1.
VH and VL were linked by a glycine-serine linker, as was the LRPAP1 epitope to VL, resulting in a VH-(GlySer)₄-VL-(GlySer)₃-LRPAP1 peptide chain. The LRPAP1 epitope was tagged with a histidine tail for subsequent detection and purification.
PCR products, amplified with these primers, were subcloned into a PCR 2.1 vector using the TOPO TA Cloning Kit (ThermoFisher Scientific, Waltham, MA) and then assembled in the pcDNA 3.1 vector. The final cloning product was used to transfect HEK 293T cells for production of bispecific constructs using X-tremeGENE HP DNA Transfection Reagent (Sigma-Aldrich Chemie GmbH, Munich, Germany).