Loss of IKK subunits limits NF-?B signaling in reovirus infected cells

Andrew J. McNamara; Pranav Danthi

. CC-BY 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. HEK293 cells were adsorbed with PBS (mock) or 10 PFU/cell of T3A. Following incubation at 37°C for 24 h, cells were treated with 10 ng/ml TNFα and incubated for 1 h. RNA was extracted from cells and IκBα gene expression was measured using RT-qPCR. Gene expression in mock infected cells treated with TNFα was set to 100%. Gene expression of each replicate, the mean value and SD are shown. *, P < 0.05 by Student's t test in comparison to mock infected cells treated with TNFα. (B) HEK293 cells were adsorbed with PBS (mock) or 10 PFU/cell of T3A. Following incubation at 37°C for 24 h, cells were treated with proteasome inhibitor PSI for 1 h, then 10 ng/ml TNFα for 30 min. Whole cell extracts were immunoblotted using antiserum specific for IKKβ, p65 Ser536 phosphorylation, p65, and PSTAIR. (C) HEK293 cells were adsorbed with PBS (mock) or 10 PFU/cell of T3A. Following incubation at 37°C for 24 h, cells were treated with 10 ng/ml TNFα and incubated for 1 h. Nuclear extracts were immunoblotted using antiserum specific for p65 or PSTAIR. (D) HEK293 cells were transfected with vectors expressing Flag-tagged IKKβ and NEMO. Following incubation at 37°C for 24 h, HEK293 cells were adsorbed with PBS (mock) or 10 PFU/cell of T3A. Following an additional incubation at 37°C for 24 h, whole cell extracts were immunoblotted using antiserum specific for FLAG, p65, p65 Ser536 phosphorylation, reovirus and PSTAIR.