Anti flag polyclonal antibody

The cDNA of porcine RIG-I were amplified from PK-15 cells and cloned into pcDNA3.1/myc-His(−)A vector (Invitrogen) to yield the Myc-tagged expression construct (Myc-RIG-I). Each of FMDV full-length viral cDNA was inserted into p3xFLAG-CMV-7.1 vector (Sigma-Aldrich) to construct plasmids expressing Flag-tagged viral proteins. A series of Flag-tagged truncated 2B constructs were generated by site-directed mutagenesis PCR. All constructed plasmids were analyzed and verified by DNA sequencing. The IFN-β promoter luciferase reporter plasmids and various hemagglutinin (HA)-tagged plasmids used in this study were kindly provided by Hongbing Shu (Wuhan University, China) (25 (link)). The commercial antibodies used in this study include: anti-Myc monoclonal antibody (Santa Cruz Biotechnology), anti-Flag monoclonal antibody (Santa Cruz Biotechnology), anti-Flag polyclonal antibody (Sigma-Aldrich), anti-RIG-I polyclonal antibody (Abcam), anti-HA tag antibody (BioLegend), anti-eukaryotic translation initiation factor 4 gamma (eIF4G) polyclonal antibody (Abcam), and anti-β-actin monoclonal antibody (Santa Cruz Biotechnology). Anti-VP1 polyclonal antibody was prepared by our laboratory (unpublished data).

The cells were lysed in SDS sample buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 50 mM DTT, 10% glycerol, and 1 mg/mL bromophenol blue). The lysate was heated to 98 °C for 10 min, and SDS-PAGE was performed to separate the proteins in the lysate. After electrophoresis, the proteins were electrotransferred onto a PVDF microporous membrane and immunodetected with the appropriate antibody. The following antibodies were used for Western blot analysis: anti-FLAG polyclonal antibody (#F7425; Sigma-Aldrich), anti-luciferase polyclonal antibody (#G7541; Promega), anti-ATF4 monoclonal antibody (#11815; Cell Signaling Technology, Beverly, MA), anti-eIF2α monoclonal antibody (#5324; Cell Signaling Technology), anti-phospho-eIF2α (Ser51) monoclonal antibody (#3398; Cell Signaling Technology), and anti-GAPDH monoclonal antibody (#2118; Cell Signaling Technology).

CD11b⁺ cells collected from the spleens of IDOL mice were treated with 20 μM MG132, 5 μg/mL LPS, and/or 20 μM caspase-1 inhibitor for 6 h, or were left untreated. The cells were lysed in SDS sample buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 50 mM DTT, 10% glycerol, and 1 mg/ml bromophenol blue). The lysate was heated to 98 °C for 10 min, and SDS-PAGE was performed to separate the proteins in the lysate. After electrophoresis, the proteins were electrotransferred onto a poly(vinylidene fluoride) microporous membrane and immunodetected with the appropriate antibody. The following antibodies were used for Western blot analysis: anti-luciferase polyclonal antibody (#G7541; Promega), and anti-FLAG polyclonal antibody (#F7425; Sigma-Aldrich). MG132 (Peptide Institute, Osaka, Japan), LPS (#L2654; Sigma-Aldrich) and caspase-1 inhibitor (#sc-3071; Santa Cruz) were obtained commercially.

The cDNA of human PABPC1 was amplified from HEK293T cells and cloned into pcDNA3.1(+)-FLAG vector (Invitrogen, Carlsbad, CA, USA) to yield the N terminal FLAG-tagged expression construct (FLAG–PABPC1). The FLAG–PABPC1 mutant (Q437N) was generated using a specific primer, as described previously [30 (link)]. The FLAG-tagged SVV 3C^pro construct and 3C^pro mutants (H48A, C160A, H48A-C160A) without protease activity were prepared in our laboratory [17 (link)]. All constructed plasmids were analyzed and verified by DNA sequencing.
The commercial antibodies used in this study included: anti-FLAG monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-FLAG polyclonal antibody (Sigma–Aldrich, St. Louis, MO, USA)anti-PABPC1 polyclonal antibody (Abcam, Cambridge, MA, USA), anti-puromycin monoclonal antibody (Merck & Co., Kenilworth, NJ, USA), and anti-β-actin monoclonal antibody (Santa Cruz Biotechnology). Anti-VP1 polyclonal antibody was prepared in our laboratory [17 (link)].

For indirect immunofluorescence experiments, anti-Flag polyclonal antibody (Sigma) and anti-Myc 9E10 mAb were used as primary antibodies. Alexa Fluor 488 goat anti-rabbit antibody (Invitrogen) and Fluorolink Cy3-labeled goat anti-mouse IgG (H + L) (GE Healthcare) were used as secondary antibodies. Cell nuclei were stained by incubation with 0.5 μg/ml Hoescht 33342 (Sigma). Slides were examined using confocal microscopy (Zeiss LSM 710) with ZEN software for the photos and standard microscopy (Zeiss Axiovert 135) with Image J software for counting the cells.

Plants overexpressing GFP-Myc, PIF1-Myc, PIF3-Myc, PIF4-Myc, PIF5-Myc, or EIN3-FLAG were grown for 4 days under the indicated conditions before cross-linking for 20 m with 1% formaldehyde under vacuum. Chromatin complexes were isolated and sonicated as described with slight modifications (Oh et al., 2009 (link)). An anti-Myc monoclonal antibody (mouse, Cell Signaling) or an anti-FLAG polyclonal antibody (rabbit, Sigma), and Protein A agarose/salmon sperm DNA (Millipore) were used for immunoprecipitation. After reverse cross-linking and protein digestion, DNA was purified using the QIAquick PCR Purification Kit (Qiagen) before being used for qPCR.