Journal: Viruses
Article Title: Porcine Respirovirus 1 Suppresses Host Type I Interferon Production and the JAK-STAT Signaling Pathway
doi: 10.3390/v15051176
Figure Lengend Snippet: The expression IFNβ and ISGs in LLC-MK2 cells infected with PRV1 (KS 17-258 strain). ( A , B ) PRV1 infection was unable to stimulate IFN-β production. LLC-MK2 cells were infected with KS 17-258 at an MOI of 1. LLC-MK2 cells infected with 100 HA units of SeV and with mock infection were used as the positive and negative control. At 24 hpi, cells were harvested with TRIzol reagent or IP lysis buffer. IFNβ expression at the mRNA level was evaluated by real-time RT-PCR with total cellular RNA. The relative mRNA expression level of IFN-β was normalized to the mRNA level of GAPDH, and the relative IFN-β mRNA level of negative control was set as 1 ( A ). The expression of two ISGs (RIG-I and ISG15) was assessed by Western blot analysis with monoclonal antibodies. F protein was detected to monitor KS 17-258 infection, and housekeeping gene GAPDH was detected as the loading control ( B ). ( C , D ) PRV1 infection suppressed IFNβ production. LLC-MK2 cells were infected with KS 17-258 at an MOI of 1 or mock-infected. At 24 hpi, cells were infected with 100 HA units of SeV to induce type I IFN production. Naïve LLC-MK2 cells were included as the negative control. After 12 h, cells were harvested with TRIzol reagent or IP lysis buffer, and IFNβ expression at the mRNA level was evaluated by real-time RT-PCR. The relative mRNA expression level of IFN-β was normalized to the mRNA level of GAPDH, and the relative IFNβ mRNA level of negative control was set as 1 ( C ). The expression of two ISGs (RIG-I and ISG15) was assessed by Western blot analysis with monoclonal antibodies. F protein was detected to monitor KS 17-258 infection, and housekeeping gene GAPDH was detected as the loading control ( D ). ( E ) PRV1 infection suppressed IFNβ production stimulated by poly I:C transfection. LLC-MK2 cells were infected with KS 17-258 at an MOI of 1 or mock-infected. At 24 hpi, cells were transfected with poly I:C at 2 μg/mL for 8 h. Naïve LLC-MK2 cells were included as the negative control. Cells were harvested with TRIzol reagent, and IFNβ expression at the mRNA level was evaluated by real-time RT-PCR. The relative mRNA expression level of IFN-β was normalized to the mRNA level of GAPDH, and the relative IFNβ mRNA level of negative control was set as 1. ***, p -value < 0.001; ****, p -value < 0.0001.
Article Snippet: A total of 1 μg RNA was used to prepare a one-step RT-PCR reaction which was formulated with 5 µL TaqMan™ Fast Virus 1-Step Master Mix (ThermoFisher Scientific, Waltham, MA, USA) and 1 μL of each predesigned primers/probe set (ThermoFisher Scientific, Waltham, MA, USA) for monkey IFNβ and GAPDH.
Techniques: Expressing, Infection, Negative Control, Lysis, Quantitative RT-PCR, Western Blot, Transfection
Thermo Fisher is a verified supplier 
Thermo Fisher manufactures this product 
