one step sybr primescript rtpcr kit  (TaKaRa)

Journal: Virologica Sinica

Article Title: Classical swine fever virus NS5A protein antagonizes innate immune response by inhibiting the NF-κB signaling.

doi: 10.1016/j.virs.2023.09.002

Figure Lengend Snippet: Fig. 4. Determination of the region of NS5A required for inhibition of the NF-κB signaling. A–C HEK293T cells in 6-well plate were transfected with full-length NS5A or NS5A truncated mutant (2.5 μg/well). After 48 h, the cells were treated with or without TNF-α for 15 min and then harvested for detection of IKKα/β phos- phorylation (A), IκBα degradation (B), quantification of IFN-α mRNA (C). The expressing level of phosphorylated IKKα/β and IκBα was analyzed by Western blotting. IFN-α mRNA was detected by RT-PCR using the One-Step SYBR Prime Script RT-PCR kit. D HEK293T cells in 24-well plate were transfected with pNF-κB-Luc reporter (1 μg/well), PRL-TK internal control (0.5 μg/well). After 48 h, the cells were treated with or without TNF-α for 15 min and the NF-κB transcriptional activity was analyzed using the Dual-Lumi Luciferase Reporter Gene Assay Kit. E The expression level of NS5A and its truncated mutants in above experiments was detected by Western blotting. Data presented as means SD. Statistical analyses were performed using GraphPad Prism version 6.01 software (GraphPad Software, La Jolla, CA, USA).**, P < 0.01; ***, P < 0.001; NS, not significant.

Article Snippet: At 48 h post-transfection, the cells were stimulated with or without TNF-α (final concentration of 20 ng/mL) for 15 min and then harvested for quantification of IFN-αmRNA using the One-Step SYBR Prime Script RTPCR kit (TaKaRa, Dalian, China) according to the manufacturer's protocol.

Techniques: Inhibition, Transfection, Mutagenesis, Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Control, Activity Assay, Luciferase, Reporter Gene Assay, Software