Anti irf3

Chromatin immunoprecipitation (ChIP) analysis was performed by using the Chromatin Immunoprecipitation (ChIP) assay kit (Millipore, 17–295) and previously described [25 (link)]. Briefly, HEK293T cells were transfected with 200 ng of each plasmid expressing either HA tagged PLpro, Flag tagged IRF3 or Flag tagged IRF3(5D) singly or in combination. At 24 hours post transfection, cells were fixed, pelleted and immediately frozen at −80°C. For immunoprecipitation the pellets were lysed and sonicated, with sonication conditions chosen to produce the desired size distribution of chromatin, between 300 and 1,200 bp. Lysates were immunoprecipitated with anti-IRF3 (Active Motif, 39033), anti-Flag (Sigma, F7425), anti-acetyl-Histone H3 (Millipore, 06–599) as a positive control, and anti-IgG (Jackson Labs, 315-005-003) as a negative control. To affirm the presence or absence of specific IRF3-binding to the IFNβ promoter following ChIP, PCR was performed. Response-specific IFNβ promoter regions were amplified by using the following primers: IFN-f 5’- GAATCCACGGATACAGAACCT-3’, IFN-r 5’-TTGACAACACGAACAGTGTCG-3’. The amplification of GAPDH served as an input control (forward 5’-CATGGGGAAGTTGAAGGTCG-3’, reverse 5’-TTGATGGTACATGACAAGGTGC-3’). PCR products were run on a 1.5% agarose gel for visualization.

The procedures used for preparation of Whole Cell Extracts (WCE), resolution by SDS-PAGE electrophoresis and immunoblot was fully detailed in⁹³ . The following primary antibodies were used in this study: anti-MKP-1/DUSP1 (M18, #sc-1102), anti-α-tubulin (B-7, #sc-5286) and anti-NFκB p65 (C-20, #sc-372) were obtained from Santa Cruz Biotechnology. Anti-phosphoT183/Y185 SAPK/JNK (G9, #9255), anti-SAPK/JNK (#9252), anti-phosphoT180/Y182 p38 MAPK (D3F9, #4511), anti-p38 MAPK (D13E1, #8690), anti-phosphoS536 NF-kappaB p65 (93H1, #3033), anti-phosphoS32 IκBα (14D4, #2859), anti-IκBα (#9242), anti-phosphoT71 ATF-2 (11G2, #5112), anti-ATF-2 (20F1, #9226), anti-phosphoS73 c-Jun (D47G9, #3270), anti-c-Jun (60A8, #9165), anti-cleaved Caspase 9 (D315, #9505) and anti-cleaved Caspase 3 (D175, #9664) were from Cell Signaling. Anti-actin clone AC-15 (#A5441) and anti-Flag M2 (#F1804) were purchased from Sigma-Aldrich, anti-IRF3 (#39033) was from Active Motif and anti-RSV (#AB1128) was from Chemicon International. Anti-phosphoS396 IRF3 was described in^{38 (link)} and anti-SeV was obtained from Dr. J. Hiscott, McGill University, Montreal, Canada. HRP-conjugated goat anti-rabbit and rabbit anti-goat (Jackson Laboratories), and goat anti-mouse (Kirkegaard & Perry Laboratories) were used as secondary antibodies. Immunoblots were quantified using the ImageQuantTL software (Molecular Dynamics).